WO2023151661A1 - Immunoconjugué et son utilisation - Google Patents

Immunoconjugué et son utilisation Download PDF

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WO2023151661A1
WO2023151661A1 PCT/CN2023/075448 CN2023075448W WO2023151661A1 WO 2023151661 A1 WO2023151661 A1 WO 2023151661A1 CN 2023075448 W CN2023075448 W CN 2023075448W WO 2023151661 A1 WO2023151661 A1 WO 2023151661A1
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seq
cancer
cdr2
cdr3
cdr1
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Chinese (zh)
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林�源
闰长青
王雪
周彩红
廖成
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江苏恒瑞医药股份有限公司
上海盛迪医药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present disclosure relates to the field of biomedicine, in particular to an immunoconjugate comprising PD-1 antibody and IL-2, a method for treating cancer, and related pharmaceutical applications.
  • Interleukin-2 also known as T cell growth factor (TCGF)
  • T cell growth factor TCGF
  • TCGF T cell growth factor
  • IL-2R IL-2 receptor
  • This model makes IL-2 a molecule that plays a central role in T cell immune responses.
  • subsequent in vivo experiments found that after knocking out IL-2 or its receptor, the animals developed autoimmunity. Subsequent experiments showed that IL-2 can activate not only effector cells such as T cells and NK cells, but also regulatory T cells (Treg), thereby suppressing excess immunity against self.
  • IL-2 acts through the IL-2R on the cell surface.
  • IL-2R includes three subunits, IL-2R ⁇ (ie CD25), IL-2R ⁇ (ie CD122) and IL-2R ⁇ (ie CD132).
  • Three subunits can form three receptor forms: high-binding receptors contain all three subunits IL-2R ⁇ / ⁇ / ⁇ , intermediate-binding receptors contain IL-2R ⁇ / ⁇ , and low-binding receptors are IL -2R ⁇ .
  • IL-2R ⁇ and IL-2R ⁇ are necessary for IL-2 to activate downstream signaling pathways.
  • IL-2 When IL-2 binds IL-2R ⁇ and IL-2R ⁇ simultaneously, the two receptor subunits form a heterodimer, and the phospho IL-2R ⁇ is not necessary for signal transduction, but it can enhance the binding of IL-2 to IL-2R ⁇ and IL-2R ⁇ .
  • Treg continuously expresses high-binding receptors IL-2R ⁇ / ⁇ / ⁇ , while unactivated CD8 + T cells and NK cells express only intermediate-binding receptors IL-2R ⁇ / ⁇ . Therefore, low-dose IL-2 preferentially binds and activates Treg, while high-dose IL-2 can also activate immune effector cells such as CD8 + T cells and NK cells.
  • high-dose IL-2 Due to its ability to activate immune effector cells, high-dose IL-2 can be used in tumor immunotherapy. However, high-dose IL-2 treatment is accompanied by serious side effects, including fever, diarrhea, and capillary leak syndrome. These side effects are associated with excessive activation of peripheral T cells and NK cells. Reducing or eliminating the activity of IL-2 on peripheral T cells and NK cells, and specifically targeting the tumor microenvironment or tumor-associated immune cells can exert its anti-tumor effect while avoiding the systemic toxicity caused by IL-2.
  • PD-1 Programmed Cell death-1 belongs to the CD28 receptor family and is an immunosuppressive receptor (Riley et al. 2009, Immunol. Rev. 29:114-25). This family also includes CD28, CTLA-4, ICOS, PD-1 and BTLA. PD-1 is mainly expressed in activated B cells, T cells and myeloid cells (Chen et al. 2013, Nat. Rev. Immunol. 13:227-42), and there are two glycoprotein ligands on the cell surface, namely PD- L1 and PD-L2.
  • PD-L1 is widely expressed in lymphocytes (such as CD4 + T cells and CD8 + T cells, macrophages, etc.), as well as peripheral tissues, various tumor cells, and virus-infected cells.
  • PD-L2 is mainly expressed on activated dendritic cells and macrophages (Dong et al. 1999, Nat. Med. 5:1365-9). After PD-1 binds to its ligand PD-L1 or PD-L2, it will down-regulate the function of T cells, including reducing the activation, differentiation and proliferation of T cells, and the secretion of cytokines.
  • PD-1 is characterized by low or no expression on the surface of peripheral T cells, but high expression on the surface of tumor-associated T cells.
  • PD-1 monoclonal antibodies such as pembrolizumab, nivolumab, and camrelizumab, have been developed for tumor immunotherapy. These monoclonal antibodies exert their medicinal effects by blocking the interaction between PD-1 and ligands in the tumor microenvironment, and have achieved good curative effects in multiple tumor types.
  • the low response rate may be due in part to low levels of tumor-infiltrating lymphocytes (Daud et al. 2016, J. Clin. Invest. 126:3447-52), and the absence of T cell activation in the tumor microenvironment (Ayers et al. 2017, J . Clin. Invest. 127:2930-2940).
  • Clinical studies have shown that the combination of PD-1 and IL-2 may have a good effect. It’s just that this IL-2 has certain systemic toxicity, such as flu-like symptoms (86.8%), rash (78.9%), fatigue (73.7%) (Diab et al. 2020, Cancer Discov.10:1158-1173), which limits The exertion of medicinal effect.
  • the present disclosure provides an immunoconjugate comprising a PD-1 antibody and IL-2 or a variant thereof that has reduced binding to a high-affinity receptor (IL-2) compared to wild-type IL-2.
  • IL-2 high-affinity receptor
  • 2R ⁇ / ⁇ / ⁇ high-affinity receptor
  • IL-2R ⁇ / ⁇ intermediate affinity receptors
  • avidity for low affinity receptors (IL-2R ⁇ ) eliminated compared to wild-type IL-2, and for Avidity is reduced more for high affinity receptors than for medium affinity receptors.
  • the immunoconjugate Due to the reduced affinity for IL-2R ⁇ / ⁇ / ⁇ and IL-2R ⁇ / ⁇ relative to wild-type IL-2, the immunoconjugate has no activity on immune cells with low or no expression of PD-1, but can It plays the role of activating immune cells with high expression of PD-1 from two aspects.
  • the PD-1 antibody blocks the interaction between PD-1/PD-L1 and PD-1/PD-L2, and relieves the immune suppression of T cells by tumors; on the other hand, the immunoconjugate binds to the surface of T cells PD-1, and only when binding to PD-1, its IL-2 variant can bind to the IL-2 receptor on the cell surface through cis (in-cis), thereby exerting the effect of IL-2 variant on PD- 1 Activation of positive T cells.
  • IL-2 variants can be used to enhance the activation and proliferation of T cells and the anti-tumor effect of PD-1 antibodies, and at the same time increase the specificity of IL-2 to PD-1 positive immune cells, avoiding the wild type Systemic toxicity is common in IL-2, which provides a more effective solution for clinical treatment of tumors.
  • the present disclosure provides PD-1 antibodies or antigen-binding fragments thereof. As well as providing IL-2 variants. And, immunoconjugates are provided comprising an antigen binding domain (e.g., a PD-1 binding domain) and a cytokine sub (e.g. IL-2 or variants thereof). And, provide PD-1 antibody or antigen-binding fragment thereof, IL-2 variant, encoding nucleic acid of immunoconjugate, vector, host cell, pharmaceutical composition, its use in the treatment or prevention of disease or disorder (such as cancer) Methods and related pharmaceutical uses.
  • an antigen binding domain e.g., a PD-1 binding domain
  • a cytokine sub e.g. IL-2 or variants thereof.
  • the present disclosure provides a PD-1 antibody or an antigen-binding fragment thereof comprising a PD-1 binding domain comprising at least one immunoglobulin single variable domain, the immunoglobulin single variable domain Domains include:
  • CDR1, CDR2 and CDR3 in any one of the amino acid sequences shown in SEQ ID NO: 3, 13-15, 17-35, 88-92, or
  • the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system.
  • the immunoglobulin single variable domain specifically binds to the PD-1 antigen or a fragment thereof.
  • the present disclosure provides a PD-1 antibody or an antigen-binding fragment thereof, which comprises a PD-1 binding domain, and the PD-1 binding domain comprises any one of the above-mentioned CDR1, CDR2 and CDR3 or any combination thereof.
  • the aforementioned PD-1 binding domain according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are respectively
  • the amino acid sequence of CDR1 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain is shown in SEQ ID NO: 7 or 93
  • the amino acid sequence of CDR2 is shown in SEQ ID NO: 8, 38 - shown in any of 40, 94-95
  • the amino acid sequence of CDR3 is shown in any of SEQ ID NO: 9, 41, 42, 96;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are shown in SEQ ID NO: 7, 38, and 41, respectively.
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are shown in SEQ ID NO: 7, 38, and 96, respectively.
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are shown in SEQ ID NO: 7, 94, and 41, respectively.
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are shown in SEQ ID NO: 93, 94, and 41, respectively.
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are shown in SEQ ID NO: 93, 94, and 96, respectively.
  • the immunoglobulin single variable domain in the aforementioned PD-1 binding domain is The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 93, 95, and 96, respectively.
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are shown in SEQ ID NO: 7, 8, and 9, respectively.
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are shown in SEQ ID NO: 7, 8, and 42, respectively.
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are shown in SEQ ID NO: 7, 38, and 6, respectively.
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are shown in SEQ ID NO: 7, 39, and 9, respectively.
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are shown in SEQ ID NO: 7, 40, and 6, respectively.
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are shown in SEQ ID NO: 7, 8, and 41, respectively.
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are shown in SEQ ID NO: 7, 8, and 42, respectively.
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are shown in SEQ ID NO: 7, 38, and 41, respectively.
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are shown in SEQ ID NO: 7, 38, and 42, respectively.
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are shown in SEQ ID NO: 7, 39, and 41, respectively.
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are shown in SEQ ID NO: 7, 39, and 42, respectively.
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are shown in SEQ ID NO: 7, 40, and 41, respectively.
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding domain are shown in SEQ ID NO: 7, 40, and 42, respectively.
  • the immunoglobulin single variable domains in the aforementioned PD-1 antibodies or antigen-binding fragments thereof are humanized, affinity matured, T cell epitopes removed, antibody deamidation reduced, and/or antibody isotropic structurally modified.
  • the immunoglobulin single variable domain is obtained through affinity maturation and has one or more changes in one or more CDRs that result in a higher affinity for PD-1 than increased in the parent.
  • the humanization process uses the heavy chain framework region IGHV3-23*01 or IGHV3-23*04 of the human germline template.
  • the immunoglobulin single variable domain in the aforementioned PD-1 binding domain comprises an amino acid sequence as shown in any one of SEQ ID NO: 3, 13-15, 17-35, 88-92, or as SEQ ID NO: 2.
  • the aforementioned PD-1 antibody or antigen-binding fragment thereof comprises or is an antibody or antigen-binding fragment thereof that specifically binds to PD-1 or a fragment thereof; Conjugated antibodies, humanized antibodies, fully human antibodies or antigen-binding fragments thereof; such antibodies or antigen-binding fragments thereof are recombinant antibodies or fragments thereof.
  • the antigen-binding fragment is a linear antibody, single chain antibody, nanobody, peptibody, domain antibody, and multispecific antibody (bispecific antibody, diabody, triabody and tetrabody, tandem di-scFv, tandem Tri-scFv).
  • At least one immunoglobulin single variable domain in the aforementioned PD-1 antibody or antigen-binding fragment thereof is VHH.
  • the aforementioned PD-1 antibody or antigen-binding fragment thereof comprises one or more (such as 2, 3, 4, 5, 6) of the aforementioned immunoglobulin single variable domains
  • the immunoglobulin Single variable domains can be the same or different and can form dimeric or multimeric molecules.
  • the aforementioned PD-1 antibody or antigen-binding fragment thereof, wherein the PD-1 binding domain comprises: the PD-1 binding domain of the heavy chain variable region (VH) and the light chain variable region (VL) .
  • the PD-1 binding domain comprises or is Fab, Fab', (Fab') 2 , ScFv, Fv, etc.
  • the PD-1 binding domain comprising a heavy chain variable region and a light chain variable region is a Fab.
  • the PD-1 antibody or antigen-binding fragment thereof comprises PD-1 binding domain 1 and PD-1 binding domain 2, or comprises PD-1 binding domain 1, or comprises PD-1 Binding domain 2.
  • the PD-1 binding domain 1 comprises at least one immunoglobulin single variable domain of any one of the foregoing.
  • the PD-1 binding domain 2 comprises a heavy chain variable region (VH) and a light chain variable region (VL) that bind to PD-1.
  • the PD-1 binding domain 2 is selected from the PD-1 binding domains in the following PD-1 antibodies: pembrolizumab, nivolumab, sintilimab (Sintilimab), Tislelizumab, Cemiplimab, Toripalimab, Camrelizumab, Penpilimab , Sasanlimab, Sasanlimab, Spartalizumab, Retifanlimab, Balstilimab, Cetrelimab, Dostarlimab (Dostarlimab; TSR-042), CS-1003, SCT-I10A, SSI-361, MEDI-0680 (AMP-514), BAT-1306, JTX-4014, JTX-4014, BI-754091, AK-103.
  • PD-1 antibodies pembrolizumab, nivolumab, sintilimab (Sintilimab), Tislelizumab, Cemi
  • the PD-1 binding domain 2 in the immunoconjugate is selected from the antigen binding domain of the following antibodies: PD-L1 antibody (for example, Durvalumab (Durvalumab), Atezol Atezolizumab, Durvalumab, Avelumab, Adebrelimab, Envafolimab, STI-A1110, CS-1001 , TQB-2450, KL-A167, IMC-001, CX-072); CTLA-4 antibodies (eg, Tremelimumab, Ipilimumab); 4-1BB antibodies (eg, Urelumab, Utomilumab).
  • PD-L1 antibody for example, Durvalumab (Durvalumab), Atezol Atezolizumab, Durvalumab, Avelumab, Adebrelimab, Envafolimab, STI-A1110, CS-1001 , TQB-2450, KL-A167, IMC-001,
  • the antigen binding domain (eg, PD-1 binding domain 2) is the portion of the antibody (eg, PD-1 antibody) that binds to an antigen (eg, PD-1), eg, its Fab', F(ab ')2, or its heavy chain variable region and light chain variable region, or the domains containing HCDR1, HCDR2, HCDR3 of its heavy chain variable region and LCDR1, LCDR2, LCDR3 of its light chain variable region.
  • the PD-1 binding domain 2 comprises pembrolizumab or an antigen-binding fragment thereof (such as Fab), for example, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in SEQ ID NO:54, 55, and 56, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 57, 58, and 59. LCDR1, LCDR2, LCDR3 showing amino acid sequences.
  • the heavy chain variable region is the heavy chain variable region in the Fab heavy chain of the amino acid sequence shown in SEQ ID NO: 52, and the light chain variable region comprises such as SEQ ID NO: 53 The light chain variable region in the Fab light chain of the amino acid sequence shown.
  • the aforementioned PD-1 antibody or antigen-binding fragment thereof further comprises a human immunoglobulin Fc region; for example, the Fc region is the Fc region of human IgG1, IgG2 or IgG4.
  • the Fc region may have mutations, exemplary mutations are L234A/L235A on IgG1, V234A/G237A/P238S/H268A/V309L/A330S/P331S on IgG2, F234A/L235A on IgG4, S228P/F234A/ L235A, N297A on IgG1, IgG2, IgG3 or IgG4, V234A/G237A on IgG2, K214T/E233P/L234V/L235A/G236 deletion/A327G/P331A/D365E/L358M on IgG1, H268Q on IgG2 /V309L/A330S /
  • Hybrid IgG2/4 Fc domains may also be used, for example an Fc having residues 117-260 from IgG2 and residues 261-447 from IgG4.
  • the Fc region of the human IgG4 has S228P, F234A, L235A and/or K447A mutations.
  • the Fc region of the human IgG1 has a L234A/L235A or L234A/L235A/P329G mutation.
  • the Fc region of human IgG1 may or may not have a C220S mutation.
  • the Fc region contained in the aforementioned PD-1 antibody or antigen-binding fragment thereof can enable the binding protein to form a dimer molecule, while prolonging the in vivo half-life of the binding protein.
  • the immunoglobulin single variable domain and the Fc region in the aforementioned PD-1 antibody or antigen-binding fragment thereof are connected through a linker.
  • the linker can be a non-functional amino acid sequence with a length of 1-20 or more amino acids and no secondary or higher structure.
  • the linker is a flexible linker, such as G 4 S, GS, GAP, (G 4 S) 2 , (G 4 S) 3 , (G 4 S) 4 , (G 4 S) 5 , ASGS, etc. .
  • the aforementioned PD-1 antibody or antigen-binding fragment thereof has at least one selected from the following The activity of:
  • the PD-1 binding K D value of the aforementioned PD-1 antibody or antigen-binding fragment thereof of the present disclosure may be ⁇ 1 ⁇ 10 -7 M, such as ⁇ 1 ⁇ 10 -8 M, or ⁇ 1 ⁇ 10 - 9 M, or ⁇ 1 ⁇ 10 -10 M.
  • the PD-1 antibody or antigen-binding fragment thereof of the present disclosure can specifically bind human PD-1 and block the interaction between PD-1 and PD-L1, and/or PD-1 and PD-L2.
  • the aforementioned PD-1 antibody or antigen-binding fragment thereof of the present disclosure is capable of inhibiting tumor growth by at least about 10%, such as at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%.
  • the aforementioned PD-1 antibody or antigen-binding fragment thereof of the present disclosure encompasses variants, for example, compared to any of SEQ ID NO: 2-3, 13-35, comprising one or more amino acid substitutions, preferably conservative Amino acid substitutions, for example, comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions, which may occur in CDR regions and/or FR regions.
  • the aforementioned PD-1 antibody or antigen-binding fragment thereof of the present disclosure is resistant to heat treatment or has high stability.
  • treatment at 40°C for up to 30 days showed no significant aggregation or degradation and was at least stable at 60°C.
  • a PD-1 antibody or antigen-binding fragment thereof that binds or competes for binding to the same epitope as the immunoglobulin single variable domain in the aforementioned PD-1 antibody or antigen-binding fragment thereof of the present disclosure.
  • a PD-1 antibody or antigen-binding fragment thereof which blocks the interaction of the immunoglobulin single variable domain and PD-1 (such as human PD) in the aforementioned PD-1 antibody or antigen-binding fragment thereof of the present disclosure.
  • PD-1 such as human PD
  • a PD-1 antibody or antigen-binding fragment thereof is provided, the binding of which to PD-1 (such as human PD-1) is controlled by the immunoglobulin in the aforementioned PD-1 antibody or antigen-binding fragment thereof of the present disclosure.
  • PD-1 such as human PD-1
  • Variable domain blockade is provided, the binding of which to PD-1 (such as human PD-1), the binding of which to PD-1 (such as human PD-1) is controlled by the immunoglobulin in the aforementioned PD-1 antibody or antigen-binding fragment thereof of the present disclosure.
  • the aforementioned PD-1 antibody or antigen-binding fragment thereof of the present disclosure reduces the binding of PD-1 to PD-L1 and/or PD-L2 by at least 60%, at least 70%, at least 80%, at least 90% , at least 95%, at least 98%.
  • the present disclosure provides a PD-1 binding protein comprising at least one of the aforementioned PD-1 binding immunoglobulin single variable domains (such as VHH).
  • At least 90% identity includes at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , at least 98%, at least 99% identity.
  • the present disclosure provides IL-2 and variants thereof.
  • the IL-2 is preferably human IL-2, and the amino acid sequence of wild-type human IL-2 is for example SEQ ID NO: 87.
  • the positions of the IL-2 variants of the present disclosure are numbered according to the wild-type IL-2 amino acid sequence shown in SEQ ID NO:87.
  • the present disclosure provides IL-2 variants that have reduced binding to high affinity receptors (IL-2R ⁇ / ⁇ / ⁇ ) and intermediate affinity receptors (IL-2R ⁇ ) compared to wild-type IL-2. / ⁇ ), and/or have a reduced affinity for the low-affinity receptor (IL-2R ⁇ ) compared to wild-type IL-2.
  • the IL-2 variant has a more reduced affinity for IL-2R ⁇ / ⁇ / ⁇ than for IL-2R ⁇ / ⁇ .
  • the affinity can be detected by conventional methods, such as ELISA, ForteBio, SPR, such as the SPR detection method provided in Example 13 of the present disclosure.
  • the present disclosure provides an IL-2 variant comprising mutation (i), mutation (ii), mutation (iii), mutation (iv) or any combination thereof, or formed by mutation (i), mutation ( ii), mutation (iii), mutation (iv) or any combination thereof.
  • the present disclosure provides mutations that confer an IL-2 variant with an altered (eg, increased or decreased) affinity for IL-2R ⁇ / ⁇ compared to wild-type IL-2.
  • the IL-2 variant has a mutation (i) that reduces affinity for IL-2R ⁇ / ⁇ compared to wild-type IL-2.
  • the IL-2 variant compared with wild-type IL-2, has a 1-1000 times, 2-800 times, 5-500 times, 5-200 times lower affinity for IL-2R ⁇ / ⁇ , 10-200 times, 10-100 times, 10-50 times, 20-200 times, 20-100 times, 5-20 times, 2-20 times, 50-100 times, 50-70 times, for example about 5, About 10, about 13, about 15, about 20, about 30, about 40, about 50, about 60, about 63, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140 , about 150, about 180, about 200, about 250, about 300, about 400, about 500 times.
  • the IL-2 variant has a 10-100 times lower affinity for IL-2R ⁇ / ⁇ than wild-type IL-2.
  • the affinity can be detected by conventional methods, such as the SPR detection method
  • the foregoing mutation (i) is located at positions 12-20, 84-95, and 126-130 of IL-2.
  • the aforementioned mutation (i) is selected from positions 12, 15, 16, 18, 19, 20, 23, 43, 69, 84, 87, 88, 91, 92, 95, 123, 126, 127, 129 , 130 or any combination thereof; in some embodiments, the mutation is selected from positions L12, E15, H16, L18, L19, D20, M23, Q22, K43, V69, D84, S87, N88, V91, I92, E95, T123, Q126, S127, I129 and S130 or any combination thereof, such as N88/D20; in some specific embodiments, the mutation is selected from 12R, 12K, 12E, 12Q, 15Q, 15R, 15A, 15S, 16N, 16A, 16E, 16D, 16G, 16S, 16T, 16V, 16P, 20A, 20H, 20Y, 20N, 20Q, 20E, 20R, 20S, 23A, 23R, 23K, 23G, 23S, 23T,
  • the present disclosure provides mutations that result in IL-2 variants that have an altered (eg, reduced) affinity for, or no binding to, IL-2R ⁇ compared to wild-type IL-2.
  • the IL-2 variant has a reduced affinity for IL-2R ⁇ compared to wild-type IL-2, or a mutation that does not bind IL-2R ⁇ (ii).
  • the affinity can be detected by conventional methods, such as the SPR detection method provided in Example 13 of the present disclosure.
  • the aforementioned mutation (ii) is selected from positions 35, 37, 38, 41, 42, 43, 45, 61, 62, 65, 68, 72 or any combination thereof; in some embodiments, the mutation selected from 38A, 38D, 38E, 62Q, 42A, 42G, 42S, 42T, 42Q, 42E, 42N, 42D, 42R, 42K, 45A, 45G, 45S, 45T, 45Q, 45E, 45N, 45D, 45R, 45K, 65R, 65E, 65K, 65H, 65Y, 65Q, 65D, 65N, 68A, 68Q, 68K, 68R, 72G, 72A, 72S, 72T, 72Q, 72E, 72N, 72D, 72R, 72K; in some embodiments, the The mutation (ii) is selected from positions K35, T37, R38, E62, P65, E68, F42, Y45, L72 or any combination thereof; in some embodiments,
  • the present disclosure provides mutations (iii) that make IL-2 variants have improved stability, expression, purity, druggability.
  • the present disclosure incorporates the IL-2 variants in WO2020125743A in its entirety.
  • the IL-2 variant has a mutation (iii) selected from positions 11, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 , 39, 40, 41, 42, 43, 44, 70, 71, 72, 78, 82, 88, 132 or any combination thereof; in some embodiments, the mutation (iii) is selected from 26Q, 29S, 30S, 71Q, 11C, 132C, 70C, 82C, 27C, 78C or any combination thereof; in some embodiments, the mutation (iii) is selected from positions N26, N29, N30, N71, Q11, L132, L70, P82, G27, F78 or any combination thereof; in some embodiments, the mutation (iii) is selected from N26Q, N29S, N30S, N71Q, Q11C/L132C, L70C/P82C, G27C/F78C or any combination thereof; in some embodiments, the Mutation (iii) is selected from
  • the IL-2 variant contains a mutation (iv) selected from position 3 and/or 125; in some embodiments, the mutation (iv) is selected from position T3 and/or C125 in some embodiments, the mutation (iv) is selected from 3A, 3G, 3Q, 3E, 3N, 3D, 3R, 3K, 3P and/or 125A, 125S, 125T, 125V mutations. In some embodiments, the mutation (iv) is selected from T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, T3P and/or C125A, C125S, C125T, C125V mutations.
  • mutation 1/mutation 2 means that both mutation 1 and mutation 2 are included.
  • IL-2 comprising or comprising only the following mutations:
  • (r) is any one of the aforementioned (a)-(h), (s) and T3A/C125A.
  • the present disclosure provides IL-2 variants comprising or having an amino acid sequence as shown in any one of SEQ ID NO: 60-68 or having at least 80%, at least 85%, at least 90% sequence identity thereto sex.
  • any of the aforementioned IL-2 variants containing mutation (i) has any one or more of 1)-5) relative to wild-type IL-2:
  • IL-2R ⁇ high affinity IL-2R receptor
  • T cells eg CD4 + and CD8 + T cells
  • any of the aforementioned IL-2 variants containing mutation (i) and mutation (ii) has any of the aforementioned 1)-5) and the following 6)-8) relative to wild-type IL-2 or more:
  • CD25 + cells eg CD25 + CD8 + T cells, Treg cells.
  • any of the aforementioned IL-2 variants containing mutation (i) and mutation (ii) and mutation (iii) and/or mutation (iv) have the aforementioned 1)-8) relative to wild-type IL-2 and any one or more of the following 9):
  • the present disclosure provides immunoconjugates comprising an antigen binding domain, and IL-2 or a variant thereof.
  • the antigen-binding domain in the immunoconjugate targets immune effector cells or tumor cells; in some specific embodiments, the immune effector cells can respond to the stimulation of IL-2 in the immunoconjugate or activated; in some specific embodiments, the immune effector cells have IL-2R ⁇ ; in some specific embodiments, the immune effector cells are, for example, NK cells, CD4 + CD25 - T cells, CD8 + CD25 - T cells.
  • the antigen binding domain in the immunoconjugate targets PD-1, CTLA-4, LAG3, TIM3, 4-1BB, OX40, GITR, CD8a, CD8b, CD4, NKp30, NKG2A, TIGIT, TGF ⁇ R1, TGF ⁇ R2, Fas, NKG2D, NKp46, PD-L1, CD107a, ICOS, TNFR2, FAP, TNC A1, TNC A2, CEA, EDB, MCSP or CD16a; in some specific embodiments, the antigen binding domain Targets PD-1 (ie, PD-1 binding domain).
  • the PD-1 binding domain is any PD-1 binding domain provided in the present disclosure.
  • the PD-1 binding domain comprises at least one immunoglobulin single variable domain
  • the immunoglobulin single variable domain comprises: SEQ ID NO: 3, 13- CDR1, CDR2 and CDR3 in any of the amino acid sequences shown in 15, 17-35, 88-92, or CDR1, CDR2 and CDR3 in any of the amino acid sequences shown in SEQ ID NO: 2, 16, the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems.
  • the PD-1 binding domain according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are respectively as SEQ ID NO: 7 , 36, 37, or as shown in SEQ ID NO: 4, 5, 6.
  • the amino acid sequence of CDR1 of the immunoglobulin single variable domain in the PD-1 binding domain is shown in SEQ ID NO: 7, and the amino acid sequence of CDR2 is shown in SEQ ID NO : 8, 38-40 shown in any one, the amino acid sequence of CDR3 is shown in any of SEQ ID NO: 9, 41, 42.
  • the immunoglobulin single variable domain comprises as shown in SEQ ID NO: 7, 36 and 37, or as shown in SEQ ID NO: 4, 5 and 6, or as shown in CDR1, CDR2 and CDR3 shown in SEQ ID NO:7, 38 and 41.
  • the amino acid sequence of the immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO: 3, 13-15, 17-35, 88-92; Or the amino acid sequence shown in any one of SEQ ID NO: 2, 16; or an amino acid sequence having at least 90% sequence identity with any of said sequences.
  • the PD-1 binding domain can also include an antigen-binding fragment targeting PD-1 other than the above-mentioned immunoglobulin single variable domain, which includes a heavy chain variable region (VH), and Light chain variable region (VL).
  • the antigen-binding fragment targeting PD-1 is selected from the PD-1 binding domain of the following PD-1 antibodies: Pembrolizumab, Nivolumab, Sindili Sintilimab, Tislelizumab, Cemiplimab, Toripalimab, Camrelizumab, Piampril Monoclonal antibody, Sepalimab, Sasanlimab, Spartalizumab, Retifanlimab, Balstilimab, Cetrelimab ), Dostarlimab (Dostarlimab; TSR-042), CS-1003, SCT-I10A, SSI-361, MEDI-0680 (AMP-514), BAT-1306, JTX-4014, J
  • the antigen-binding domain in the immunoconjugate is selected from the antigen-binding domain of the following antibodies: PD-L1 antibodies (for example, durvalumab (Durvalumab), atezolizumab (Atezolizumab), Durvalumab, Avelumab, Adebrelimab, Envafolimab, STI-A1110, CS-1001, TQB -2450, KL-A167, IMC-001, CX-072); CTLA-4 antibodies (eg, Tremelimumab, Ipilimumab); 4-1BB antibodies (eg, Uri Urelumab, Utomilumab).
  • PD-L1 antibodies for example, durvalumab (Durvalumab), atezolizumab (Atezolizumab), Durvalumab, Avelumab, Adebrelimab, Envafolimab, STI-A1110, CS-1001, TQB
  • the antigen binding domain (eg, PD-1 binding domain) is the part of the antibody (eg, PD-1 antibody) that binds to an antigen (eg, PD-1), eg, its Fab', F(ab' ) 2, or its heavy chain variable region and light chain variable region, or a domain containing HCDR1, HCDR2, HCDR3 of its heavy chain variable region and LCDR1, LCDR2, LCDR3 of its light chain variable region.
  • the PD-1 binding domain comprises pembrolizumab or an antigen-binding fragment thereof (e.g., Fab), e.g., comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein , the heavy chain variable region comprises HCDR1, HCDR2, HCDR3 of the amino acid sequence shown in SEQ ID NO: 54, 55, 56, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 57, 58, 59 Amino acid sequence LCDR1, LCDR2, LCDR3.
  • Fab antigen-binding fragment thereof
  • the heavy chain variable region is the heavy chain variable region in the Fab heavy chain of the amino acid sequence shown in SEQ ID NO: 52, and the light chain variable region comprises such as SEQ ID NO: 53 The light chain variable region in the Fab light chain of the amino acid sequence shown.
  • the immunoconjugate comprises any of the following:
  • PD-1 binding domain 1 and IL-2 or variants thereof; wherein, the PD-1 binding domain 1 comprises at least one single variable domain of any immunoglobulin provided by the present disclosure
  • the PD-1 binding domain 1 comprises at least one immunoglobulin single protein provided by the present disclosure variable domain;
  • the PD-1 binding domain 2 is any PD-1 binding domain comprising VH and VL provided by the present disclosure.
  • the IL-2 or variant thereof in the immunoconjugate is any IL-2 or variant thereof provided in the foregoing disclosure.
  • the IL-2 is wild-type human IL-2.
  • the IL-2 variant comprises the aforementioned mutation (i), mutation (ii), mutation (iii), mutation (iv) or any combination thereof provided by the present disclosure, or the mutation (i), mutation (ii), mutation (iii), mutation (iv) or any combination thereof.
  • the IL-2 variant comprises or only comprises the mutation of any one of (a)-(r) and (s) provided in the present disclosure.
  • the IL-2 variant contains an amino acid sequence as shown in any one of SEQ ID NO: 60-68 or has at least 80%, at least 85%, at least 90% identity therewith Sexual sequence, or consists of the above sequence.
  • the immunoconjugate further comprises a human immunoglobulin Fc region, such as the Fc region of human IgG1, IgG2, IgG3 or IgG4.
  • the antigen binding domain (such as PD-1 binding structure) in the immunoconjugate Domain) is located at the N-terminus of the Fc region, and the IL-2 or variant thereof is located at the N-terminus or C-terminus of the Fc region. In some specific embodiments, the antigen-binding domain is located at the N-terminal of the Fc, and the IL-2 or variant thereof is located at the C-terminal of the Fc region.
  • the valence ratio of the antigen binding domain (such as PD-1 binding domain) to IL-2 or its variants in the immunoconjugate is 6:1 to 1:3 (such as 4: 1 to 1:2), for example 4:1, 2:1, 3:1 or 1:1.
  • the valence ratio of the PD-1 binding domain to IL-2 or its variant refers to the ratio of the included PD-1 binding domain to IL-2 or its variant in an immunoconjugate molecule. body ratio.
  • the valence ratio is the number ratio of PD-1 binding domain 1 comprising at least one immunoglobulin single variable domain to IL-2 or a variant thereof.
  • the valency ratio is the ratio of the sum of the numbers of PD-1 binding domain 1 and PD-1 binding domain 2 to the number of IL-2 or a variant thereof.
  • one immunoconjugate contains 1 or 2 IL-2 or its variants, and contains 1, 2, 3, 4, 5 or 6 antigen binding domains (such as PD-1 binding domain).
  • the immunoconjugate further comprises an Fc region comprising a knob-into-hole modification of the first subunit Fc1 and the second subunit Fc2 capable of associating with each other.
  • amino acid mutations are included in Fc1 and Fc2 such that Fc1 preferentially pairs with Fc2 or preferentially forms a heterodimer over Fc1.
  • the mutation is located in CH3 of Fcl and Fc2.
  • amino acid mutations in Fcl and Fc2 result in greater electrostatic complementarity than wild type without the mutation.
  • amino acid mutations in Fcl and Fc2 result in greater steric complementarity than wild type without the mutation.
  • one or more amino acid residues in the CH3 domain of Fc1 are mutated with one or more amino acid residues having a greater side chain volume in Fc1 and Fc2, e.g., within the CH3/CH3 interface, thereby One or more, preferably two or three amino acid residues in the CH3 domain of Fc2 interacting with the CH3 domain of Fc1 are generated on the surface of the CH3 domain of Fc1 (or knob, Knob), with smaller side chains Bulk amino acid residues are mutated to create a depression (or hole, Hole) on the surface of the CH3 domain of Fc2 that interacts with the CH3 domain of Fcl.
  • the import residue with greater side chain volume is phenylalanine (F), tyrosine (Y), arginine (R) or tryptophan (W).
  • the import residue with smaller side chain volume is serine (S), alanine (A), valine (V) or threonine (T).
  • the Fc1 contains at least one or at least two amino acid mutations selected from T366, L368 and Y407 (hole mutation modification, also known as Hole modification), and the Fc2 contains T366 (knob mutation modification, also known as Hole modification). called Knok modification); or the Fc1 contains T366 (knob mutation modification), and the Fc2 contains at least one or at least two amino acid mutations selected from T366, L368 and Y407 (hole mutation modification).
  • the Fc1 contains at least one or at least two amino acid mutations selected from T366S, L368A and Y407V (knob mutation modification), the Fc2 contains T366W (knob mutation modification); or the Fc1 contains T366W (knob mutation modification), and the Fc2 contains at least one or at least two amino acid mutations selected from T366S, L368A and Y407V (knob mutation modification).
  • the IL-2 or its variants are located in the polypeptide chain where Fc1 is located.
  • Fc1 and Fc2, such as CH3 may contain a natural non-cysteine to cysteine mutation, such as S354C in Fc1 and Y349C in Fc2; or Y349C in Fc1 and Y349C in Fc2 Contains S354C.
  • Fc1 and Fc2 for example, within the Fc1CH3/Fc2CH3 interface, comprise the following amino acid mutations or combinations thereof:
  • Fc1 and Fc2 further comprise amino acid mutations that allow the formation of an electrostatic interaction interface between Fc1 and Fc2 (eg, CH3 and CH3).
  • Amino acid mutations forming an electrostatic interaction interface are for example selected from the following:
  • Fc1 and/or Fc2 comprise domains from different antibody subtypes, such as CH3 from different antibody subtypes.
  • Fc1 and/or Fc2, eg, CH3, comprise amino acid mutations for altering effector function.
  • antibody effector functions include, but are not limited to: C1q binding and complement-dependent cytotoxicity, Fc receptor (e.g., Fc ⁇ R) binding, antibody-dependent cytotoxicity (ADCC), phagocytosis, cell surface receptors (e.g., B cell receptor) and B cell activation.
  • Amino acid mutations that alter effector function are, for example, selected from the following:
  • the Fc1 and/or the Fc2 comprise mutations that reduce effector function (eg, ADCC), eg, L234A/L235A, L234A/L235A/P329G.
  • ADCC effector function
  • Fcl and Fc2, eg, in CH3 thereof comprise amino acid mutations for altering half-life (eg, extending half-life).
  • the half-life is dependent on FcRn binding affinity. An increase in half-life may allow for a reduction in the amount of drug administered to a patient, and/or less frequent dosing.
  • the Fc region has M252Y, S254T and/or T256E mutations.
  • the immunoconjugate is PEGylated or conjugated/fused to (human serum) albumin, albumin binding protein.
  • Fc1 and/or Fc2, eg, CH3, comprise one or more isoallotypic mutations.
  • the heteroallotypic mutations are D356E and L358M.
  • Fc1 and/or Fc2 comprise a C220 mutation, such as a C220S mutation.
  • the immunoconjugate comprises a polypeptide chain combination of the amino acid sequences shown below: SEQ ID NO: 73 and 74; SEQ ID NO: 75 and 76; SEQ ID NO: 77 and 78; SEQ ID NO: 79 and 80; SEQ ID NO:43 and 44; SEQ ID NO:45 and 46; SEQ ID NO:47, 44 and 48; SEQ ID NO:47, 46 and 48; SEQ ID NO:49, 50 and 48; SEQ ID NO:47, 44 and 48; SEQ ID NO:49, 50 and 48; NOs: 51 and 46; SEQ ID NOs: 102 and 103 or combinations with any of the above polypeptide chains have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% , an amino acid sequence of at least 99% identity.
  • Embodiment 1 An immunoconjugate comprising:
  • IL-2R ⁇ / ⁇ / ⁇ high-affinity receptors
  • IL-2R ⁇ / ⁇ high-affinity receptors
  • IL-2R ⁇ / ⁇ medium-affinity receptors
  • the reduction is even greater; for example, the IL-2 variant contains a mutation selected from N88D or D20A.
  • Embodiment 2 The immunoconjugate of embodiment 1, wherein the IL-2 variant comprises a mutation selected from F42, Y45, L72 or any combination thereof;
  • it contains a mutation selected from F42A, Y45A, L72G or any combination thereof;
  • F42A/L72G contains F42A/Y45A, Y45A/L72G, F42A/Y45A/L72G mutations.
  • Embodiment 3 The immunoconjugate of embodiment 1 or 2, wherein the IL-2 variant comprises a mutation selected from N26Q, N29S, N30S, N71Q, Q11C and L132C, L70C and P82C, G27C and F78C or any combination thereof;
  • it contains a mutation or combination selected from:
  • Embodiment 4 An immunoconjugate comprising:
  • IL-2 an antigen binding domain, and IL-2, or a variant thereof, said IL-2 variant comprising a mutation of the first type that renders the IL-2 variant a reduced affinity for the wild-type IL-2 Avidity for intermediate affinity receptors (IL-2R ⁇ / ⁇ );
  • said first type of mutation is selected from: L12, E15, H16, L18, L19, D20, M23, Q22, K43, V69, D84, S87, N88, V91, I92, E95, T123, Q126, S127, I129 and S130;
  • the first type of mutation is selected from: L12R, L12K, L12E, L12Q, E15Q, E15R, E15A, E15S, H16A, H16E, H16D, H16G, H16S, H16T, H16V, H16P, D20A, D20H, D20Y, D20N, M23A, M23R, M23K, M23G, M23S, M23T, M23V, M23P, S87K, S87A, D84S, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, D84G, D84A, D84T, D84P, N88D, N8 8A, N88S, N88T, N88R, N88I, V91A, V91T, V91E, I92A, E95S, E95A, E95R, E95Q, E95G, E95T, E95V, E95P, E
  • the first type of mutation is selected from: N88D, N88R, D20A, D20N, V91T, Q126D, H16A/D84S;
  • the first type of mutation is selected from N88D or D20A.
  • Embodiment 5 The immunoconjugate of embodiment 4, wherein the IL-2 variant contains a second type of mutation that renders the IL-2 variant with reduced IL-2 compared to wild-type IL-2 or abrogated affinity for high-affinity receptors (IL-2R ⁇ / ⁇ / ⁇ );
  • the second type of mutation is selected from R38, E62, P65, E68, F42, Y45, L72 or any combination thereof;
  • the second type of mutation is selected from R38A, R38D, R38E, E62Q, P65R, P65E, P65K, P65H, P65Y, P65Q, P65D, P65N, E68A, E68Q, E68K, E68R, F42A, F42G, F42S, F42T , F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, L72K;
  • the second type of mutation is selected from F42A, Y45A, L72G, F42A/L72G, F42A/Y45A, Y45A/L72G or F42A/Y45A/L72G.
  • Embodiment 6 The immunoconjugate of embodiment 4 or 5, wherein the IL-2 variant contains a third type of mutation that renders the IL-2 variant more potent than wild-type IL-2. 2 increased stability;
  • the third type of mutation is selected from N26, N29, N30, N71, Q11, L132, L70, P82, G27, F78 or any combination thereof;
  • the third type of mutation is selected from N26Q, N29S, N30S, N71Q, Q11C/L132C, L70C/P82C, G27C/F78C or any combination thereof;
  • the third type of mutation is selected from: N26Q, N29S, N30S, N26Q/N29S/N71Q, N26Q/N29S, N26Q/N30S, N26Q/N30S/Q11C/L132C, N26Q/N29S/L70C/P82C, N26Q/ N30S/G27C/F78C.
  • Embodiment 7 An immunoconjugate comprising: a PD-1 binding domain, and IL-2 or a variant thereof;
  • the PD-1 binding domain comprises at least one immunoglobulin single variable domain, and the immunoglobulin single variable domain comprises:
  • CDR1, CDR2 and CDR3 in any of the amino acid sequences shown in SEQ ID NO: 3, 13-15, 17-35, or
  • the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system;
  • the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are respectively
  • amino acid sequence of CDR1 of the immunoglobulin single variable domain is shown in SEQ ID NO: 7
  • amino acid sequence of CDR2 is shown in any of SEQ ID NO: 8, 38-40
  • amino acid sequence of CDR3 As shown in any of SEQ ID NO: 9, 41, 42;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NO: 7, 38, and 41, respectively.
  • Embodiment 8 The immunoconjugate according to any one of embodiments 1 to 7, wherein the IL-2 variant comprises a combination of mutations selected from group 1) and group 2):
  • Group 1) mutations are selected from: F42A/L72G, F42A/Y45A, Y45A/L72G and F42A/Y45A/L72G;
  • Group mutations are selected from: N88R, N88G, N88I, N88D, D20H, D20Y, V91T, Q126L, Q126D, H16A/D84S;
  • the IL-2 variant contains the following combination of mutations: F42A/L72G/N88D, F42A/L72G/N88R, F42A/L72G/D20A, F42A/L72G/D20N, F42A/L72G/V91T, F42A/L72G/Q126D, F42A/L72G/H16A/D84S.
  • Embodiment 9 The immunoconjugate of embodiment 8, wherein the IL-2 variant further comprises a group 3) mutation selected from the group consisting of: N26Q, N29S, N30S, N26Q /N29S/N71Q, N26Q/N29S, N26Q/N30S, N26Q/N30S/Q11C/L132C, N26Q/N29S/L70C/P82C and N26Q/N30S/G27C/F78C;
  • the IL-2 variant contains the following combination of mutations:
  • Embodiment 10 The immunoconjugate of any one of embodiments 1 to 9, wherein the IL-2 variant contains a mutation selected from T3 and/or C125 or a combination thereof;
  • T3A and/or C125A, C125S, C125T, C125V mutations are examples.
  • Embodiment 11 The immunoconjugate of any one of embodiments 1 to 10, said IL-2 variant having an amino acid sequence as set forth in any one of SEQ ID NOs: 60-68.
  • Embodiment 12 In the immunoconjugate of any one of embodiments 4 to 6, 8 to 10,
  • the antigen is selected from:
  • PD-1 PD-1, CTLA-4, LAG3, TIM3, 4-1BB, OX40, GITR, CD8a, CD8b, CD4, NKp30, NKG2A, TIGIT, TGF ⁇ R1, TGF ⁇ R2, Fas, NKG2D, NKp46, PD-L1, CD107a, ICOS, TNFR2, FAP, TNC A1, TNC A2, CEA, EDB, MCSP, or CD16a;
  • Immune checkpoint antigens such as PD-1, PD-L1, CTLA-4;
  • the antigen binding domain comprises a VH domain and a VL domain of an antibody selected from the following, or HCDR1, HCDR2, HCDR3 of VH and LCDR1, LCDR2, LCDR3 of VL:
  • PD-1 antibodies eg, Pembrolizumab, Nivolumab, Sintilimab, Tislelizumab, Cemiplimab ), Toripalimab (Toripalimab), Camrelizumab (Camrelizumab), Pembrolizumab, Sepalimab, Sasanlimab (Sasanlimab), Spartakizumab ( Spartalizumab), Retifanlimab, Balstilimab, Cetrelimab, Dostarlimab (TSR-042), CS-1003, SCT-I10A, SSI -361, MEDI-0680 (AMP-514), BAT-1306, JTX-4014, JTX-4014, BI-754091, AK-103);
  • CTLA-4 antibodies eg, Tremelimumab, Ipilimumab
  • 4-1BB antibodies eg, Urelumab, Utomilumab
  • PD-L1 antibodies eg, Durvalumab, Atezolizumab, Durvalumab, Avelumab, Adebelizumab ( Adebrelimab), envafolimab, STI-A1110, CS-1001, TQB-2450, KL-A167, IMC-001, CX-072).
  • Embodiment 13 The immunoconjugate according to any one of embodiments 1 to 3, 12, wherein the PD-1 binding domain comprises at least one single variable domain of an immunoglobulin that can be Variable domains include:
  • CDR1, CDR2 and CDR3 in any one of the amino acid sequences shown in SEQ ID NO: 3, 13-15, 17-35, 88-92, or
  • the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system;
  • the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are respectively
  • amino acid sequence of CDR1 of the immunoglobulin single variable domain is shown in SEQ ID NO: 7 or 93
  • the amino acid sequence of CDR2 is shown in any one of SEQ ID NO: 8, 38-40, or 94-95.
  • amino acid sequence of CDR3 is shown in any one of SEQ ID NO: 9, 41, 42, 96;
  • immunoglobulin single variable domain is selected from any of the following:
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NO: 7, 38, and 41, respectively.
  • Embodiment 14 The immunoconjugate of embodiment 7 or 13, wherein the immunoglobulin single variable domain is:
  • Camelid antibodies chimeric antibodies, humanized antibodies, fully human antibodies or antigen-binding fragments thereof.
  • Embodiment 15 The immunoconjugate of embodiment 13 or 14, wherein the immunoglobulin single variable domain comprises
  • Embodiment 16 The immunoconjugate according to any one of embodiments 1 to 3, 12, wherein the PD-1 binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein, the heavy chain variable region comprises HCDR1, HCDR2, HCDR3 of the amino acid sequence shown in SEQ ID NO: 54, 55, 56, and the light chain variable region comprises SEQ ID NO: 57, 58, 59 LCDR1, LCDR2, LCDR3 showing the amino acid sequence;
  • VH heavy chain variable region
  • VL light chain variable region
  • the heavy chain variable region is the heavy chain variable region in the Fab heavy chain of the amino acid sequence shown in SEQ ID NO: 52, and the light chain variable region comprises amino acids shown in SEQ ID NO: 53 The light chain variable region in the Fab light chain of the sequence.
  • Embodiment 17 The immunoconjugate of any one of the preceding embodiments, further comprising a human immunoglobulin Fc region; for example, the Fc region is the Fc region of a human IgGl, IgG2, IgG3 or IgG4.
  • Embodiment 18 The immunoconjugate of embodiment 17, wherein the Fc region of human IgG1 contains one or more amino acids that reduce binding to Fc receptors (FcR, such as Fc ⁇ R), and/or reduce effector function mutation;
  • FcR Fc receptors
  • the Fc region of human IgG1 contains one or more amino acid mutations that reduce the binding to Fc ⁇ receptor (Fc ⁇ R), and/or reduce the effect of ADCC;
  • the Fc region of the human IgG1 contains mutations of L234, L235, P329 or any combination thereof;
  • the Fc region of the human IgG1 contains L234A/L235A or L234A/L235A/P329G mutations Change.
  • Embodiment 19 The immunoconjugate of embodiment 17 or 18, said Fc region comprising a knob-into-hole modification that promotes association (or heterodimerization) of the first and second subunits of the Fc region;
  • the knob-into-hole modification means, for example, that amino acid residues in the CH3 region of the first subunit of the Fc region are replaced with amino acid residues having a larger side chain volume, so as to replace A bulge is created in the CH3 region of one subunit that can be placed in a cavity in the CH3 region of a second subunit, and amino acid residues in the CH3 region of the second subunit of the Fc region are replaced with more Substitution of amino acid residues with a small side chain volume to create a cavity in the CH3 region of the second subunit, where a bulge in the CH3 region of the first subunit can be placed; as another example, said amino acid having a larger side chain volume
  • the residues are selected from R, F, Y, W, and the amino acid residues with smaller side chain volumes are selected from A, S, T, V.
  • Embodiment 20 The immunoconjugate of embodiment 19, wherein
  • the first subunit of the Fc region contains a T366 mutation, and the second subunit contains a mutation selected from T366, L368, Y407 or any combination thereof;
  • the first subunit of the Fc region contains a S354 or E356 mutation, and the second subunit contains a Y349 mutation; or
  • the first subunit of the Fc region contains S354 and T366 mutations, and the second subunit contains Y349, T366, L368 and Y407 mutations;
  • the first subunit of the Fc region contains a T366W mutation, and the second subunit contains a mutation selected from T366S, L368A, Y407V or any combination thereof;
  • the first subunit of the Fc region contains a S354C or E356C mutation, and the second subunit contains a Y349C mutation; or
  • the first subunit of the Fc region contains the S354C/T366W mutation, and the second subunit contains the Y349C/T366S/L368A/Y407V mutation.
  • Embodiment 21 The immunoconjugate according to embodiment 19 or 20, wherein the second subunit of the Fc region further contains H435 and/or R436 mutations, preferably H435R/Y436F, H435R, H435K mutations.
  • Embodiment 22 The immunoconjugate of any one of embodiments 17 to 21, wherein the Fc region of human IgG1 contains mutations that enhance FcRn-mediated recycling, and/or extend half-life;
  • the Fc region of the human IgG1 contains M252 and/or M428 mutations
  • the Fc region of human IgG1 contains M252Y/M428V, M252Y/M428L mutations.
  • Embodiment 23 The immunoconjugate according to any one of embodiments 17 to 22, wherein the first subunit and/or the second subunit of the Fc region of human IgG1 contains a C220 mutation, such as a C220S mutation.
  • Embodiment 24 The immunoconjugate according to any one of the preceding embodiments, the PD-1 binding domain or antigen binding domain is located at the N-terminus of the Fc region, and the IL-2 variant is located at the N-terminal of the Fc region. N-terminal or C-terminal;
  • the PD-1 binding domain or antigen binding domain is located at the N-terminal of Fc, and the IL-2 variable The body is located at the C-terminus of the Fc region.
  • Embodiment 25 The immunoconjugate according to any one of the preceding embodiments, wherein the valency ratio of the PD-1 binding domain or antigen binding domain to IL-2 or a variant thereof is 6:1 to 1 :3 (eg 4:1 to 1:2), preferably 4:1, 2:1, 3:1 or 1:1; or, one immunoconjugate contains 1 or 2 IL-2 or Its variants contain 1, 2, 3, 4, 5 or 6 PD-1 binding domains or antigen binding domains.
  • Embodiment 26 The immunoconjugate according to any one of embodiments 1 to 3, 7, 11 to 25, comprising the polypeptide chain combination shown in any one of I)-VI) below:
  • the first polypeptide chain [PD-1 binding domain 1]-[the first subunit of the Fc region]-[linker 1]-[IL-2 or its variants],
  • the second polypeptide chain [PD-1 binding domain 1] - [the second subunit of the Fc region];
  • the second polypeptide chain [PD-1 binding domain 1] - [linker 2] - [PD-1 binding domain 1] - [the second subunit of the Fc region];
  • the first polypeptide chain [Fab heavy chain of PD-1 binding domain 2] - [the first subunit of the Fc region] - [linker 1] - [IL-2 or its variants],
  • the second polypeptide chain [PD-1 binding domain 1] - [the second subunit of the Fc region],
  • the third polypeptide chain [Fab light chain of PD-1 binding domain 2];
  • the first polypeptide chain [Fab heavy chain of PD-1 binding domain 2] - [the first subunit of the Fc region] - [linker 1] - [IL-2 or its variants],
  • the second polypeptide chain [PD-1 binding domain 1] - [linker 2] - [PD-1 binding domain 1] - [the second subunit of the Fc region],
  • the third polypeptide chain [Fab light chain of PD-1 binding domain 2];
  • the second polypeptide chain [PD-1 binding domain 1] - [Linker 2] - [Fab light chain of PD-1 binding domain 2] - [the second subunit of Fc region],
  • the third polypeptide chain [Fab light chain of PD-1 binding domain 2];
  • the second polypeptide chain [PD-1 binding domain 1] - [linker 2] - [PD-1 binding domain 1] - [the second subunit of the Fc region];
  • linker 1 is selected from embodiments 11 to Any PD-1 binding domain in 13;
  • linker 2 is selected from any PD-1 binding domain in embodiment 14;
  • the linker is a (G x S) y linker, wherein x is selected from an integer of 1-5, and y is selected from an integer of 1-6. Integer; and for example, linker 1 is (G 4 S) 3 (SEQ ID NO: 104), linker 2 is (G 4 S) 2 (SEQ ID NO: 105), linker 3 is G 4 S (SEQ ID NO: 105) ID NO: 106).
  • linkers include, but are not limited to, such as (G m S n ) h or (GGNGT) h (SEQ ID NO: 107) or (YGNGT) h (SEQ ID NO: 108) or (EPKSS) h (SEQ ID NO : 109), wherein m, n are each independently selected from an integer of 1-8 (for example, 1, 2, 3, 4, 5, 6, 7 or 8), and h is independently selected from 1-8 An integer of 20 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).
  • the order of the above polypeptide chains is from N-terminus to C-terminus.
  • Embodiment 27 The immunoconjugate of any one of the preceding embodiments, comprising a polypeptide chain combination of amino acid sequences as shown below:
  • amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any combination of the above polypeptide chains.
  • the numbering rules for the above loci are the Kabat EU index.
  • the present disclosure provides a fusion protein comprising or being any of the aforementioned immunoconjugates.
  • the present disclosure provides polynucleotides encoding PD-1 antibodies or antigen-binding fragments thereof, IL-2 variants, immunoconjugates of the present disclosure.
  • a nucleic acid of the present disclosure may be RNA, DNA or cDNA. According to some embodiments of the present disclosure, the nucleic acids of the present disclosure are substantially isolated nucleic acids.
  • a nucleic acid of the disclosure can also be in the form of, can be present in, and/or can be part of a vector, such as a plasmid, cosmid, YAC, or viral vector.
  • the vector may especially be an expression vector, ie a vector providing for in vitro and/or in vivo (ie in a suitable host cell, host organism and/or expression system) expression of a PD-1 antibody or antigen-binding fragment thereof.
  • the expression vector typically comprises at least one nucleic acid of the present disclosure operably linked to one or more suitable expression control elements (eg, promoters, enhancers, terminators, etc.). The selection of said elements and their sequences for expression in a particular host is within the general knowledge of the person skilled in the art. right Regulatory elements and other elements useful or necessary for the expression of the PD-1 antibody or antigen-binding fragment thereof of the present disclosure are, for example, promoters, enhancers, terminators, integrators, selectable markers, leader sequences, and reporter
  • Nucleic acids of the disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and/or recombinant DNA techniques) based on the amino acid sequence information of the polypeptides of the disclosure, and/or can be isolated from suitable natural sources.
  • the present disclosure provides recombinant host cells expressing or capable of expressing one or more PD-1 antibodies or antigen-binding fragments thereof, IL-2 variants, immunoconjugates of the present disclosure and/or containing nucleic acids or vectors of the present disclosure.
  • the host cell is a bacterial cell, a fungal cell, or a mammalian cell.
  • Bacterial cells include, for example, Gram-negative bacterial strains (such as Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus spp. (Bacillus) strains, Streptomyces (Streptomyces) strains, Staphylococcus (Staphylococcus) strains and Lactococcus (Lactococcus) strains) cells.
  • Gram-negative bacterial strains such as Escherichia coli strains, Proteus strains, and Pseudomonas strains
  • Gram-positive bacterial strains such as Bacillus spp. (Bacillus) strains, Streptomyces (Streptomyces) strains, Staphylococcus (Staphylococcus) strains and Lactococcus (Lactococcus) strains
  • Fungal cells include, for example, cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or Saccharomyces (such as Saccharomyces cerevisiae), fission yeast Schizosaccharomyces (such as Schizosaccharomyces pombe), Pichia (such as Pichia pastoris and Pichia methanolica) and Hansen A cell of a species of Saccharomyces (Hansenula).
  • Saccharomyces such as Saccharomyces cerevisiae
  • fission yeast Schizosaccharomyces such as Schizosaccharomyces pombe
  • Pichia such as Pichia pastoris and Pichia methanolica
  • Hansen A cell of a species of Saccharomyces Hansenula
  • Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.
  • amphibian cells insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins may also be used in the present disclosure.
  • the present disclosure provides a method for preparing PD-1 antibody or its antigen-binding fragment, IL-2 variant, and immunoconjugate, comprising: expressing the target protein in the aforementioned host cell, and automatically The protein of interest is isolated from the host cells.
  • a purification step may also be included, for example, purify with an A or G Sepharose FF column containing an adjusted buffer to wash away non-specifically bound components, then elute the bound antibody with a pH gradient method, and use SDS-PAGE detection, collection.
  • concentration by filtration is performed using conventional methods. Soluble mixtures and aggregates can also be removed by conventional methods such as molecular sieves and ion exchange.
  • the obtained product needs to be immediately frozen, such as -70°C, or freeze-dried.
  • Antibodies or antigen-binding fragments engineered in the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • Mammalian-like expression systems lead to glycosylation of antibodies, especially is the highly conserved N-terminus of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free medium in bioreactors for antibody production.
  • the culture fluid that secretes the antibody can be purified and collected using conventional techniques.
  • Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and aggregates can also be removed by conventional methods such as molecular sieves and ion exchange.
  • compositions comprising the foregoing PD-1 antibodies or antigen-binding fragments thereof, IL-2 variants and/or immunoconjugates.
  • a pharmaceutical composition which contains an effective amount of the above-mentioned PD-1 antibody or antigen-binding fragment thereof, an immunoconjugate, and at least one pharmaceutically acceptable excipient, diluted or carrier.
  • the unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of PD-1 antibody or antigen-binding fragment thereof, IL-2 variant and/or immunoconjugate, or a pharmaceutical composition
  • the amount of PD-1 antibody or antigen-binding fragment thereof, IL-2 variant and/or immunoconjugate in a unit dose is 0.1-2000 mg, and in some embodiments, 1-1000 mg.
  • an article or product comprising the aforementioned PD-1 antibody or antigen-binding fragment thereof, IL-2 variant and/or immunoconjugate.
  • the article comprises a container and a label.
  • Containers such as bottles, syringes and test tubes.
  • the container contains a composition effective for treating a condition.
  • the label on or associated with the container indicates that the composition is used to treat the condition of choice.
  • the composition contains the aforementioned PD-1 antibody or its antigen-binding fragment, and immunoconjugate.
  • composition is provided, and it contains:
  • composition when it is a pharmaceutical composition, it also contains at least one pharmaceutically acceptable excipient, diluent or carrier.
  • the present disclosure provides the use of the aforementioned PD-1 antibody or its antigen-binding fragment, IL-2 variant, immunoconjugate, polynucleotide, composition (including pharmaceutical composition) in at least one of the following:
  • CTL cytotoxic T cells
  • CTL cytotoxic T cells
  • Cytotoxic T cells (CTL) for high expression of PD-1 antibody have higher cell activation efficiency than cytotoxic T cells (CTL) with low or no expression of PD-1 antibody;
  • CD4 + and/or CD8 + T for high expression of PD-1 antibody, with lower expression than PD-1 antibody Cell proliferation efficiency of CD4 + and/or CD8 + T cells expressing or not.
  • the present disclosure provides the aforementioned PD-1 antibodies or antigen-binding fragments thereof, IL-2 variants, immunoconjugates, polynucleotides, compositions (including pharmaceutical compositions) for treating, alleviating, preventing, and diagnosing diseases or disorders method.
  • a method for improving, alleviating, treating or preventing a disease comprising administering to a subject an effective amount of the aforementioned PD-1 antibody or an antigen-binding fragment thereof, an immunoconjugate, a multinuclear Nucleotides, compositions (including pharmaceutical compositions).
  • the use of the PD-1 antibody or its antigen-binding fragment, IL-2 variant, and immunoconjugate of the present disclosure for preparing a medicament for improving, alleviating, treating or preventing a disease is provided.
  • the aforementioned disease is a proliferative disorder or any other disease or disorder characterized by uncontrolled cell growth (such as cancer, cancer and tumor are used interchangeably in this disclosure), such as PD-L1 overexpression Related related diseases, or related diseases that benefit from the treatment of PD-1 antibodies or antigen-binding fragments thereof, such as cancer.
  • the aforementioned cancer is a solid tumor or a hematological tumor.
  • the aforementioned cancer is advanced or metastatic.
  • the aforementioned cancer is selected from the following or a combination thereof: lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer , liver cancer, melanoma, kidney cancer, squamous cell carcinoma, hematologic cancer, or any other disease or condition characterized by uncontrolled cell growth.
  • liver cancer HCC
  • colorectal cancer ovarian cancer
  • endometrial cancer breast cancer
  • triple negative breast cancer pancreatic cancer
  • gastric (stomach/gastric) cancer cervical cancer
  • head and neck cancer thyroid cancer Carcinoma
  • testicular cancer urothelial carcinoma
  • lung cancer small cell lung cancer, non-small cell lung cancer
  • melanoma non-melanoma skin cancer (squamous and basal cell carcinoma)
  • glioma glioma
  • renal cancer RCC
  • lymphoma NHL or HL
  • acute myelogenous leukemia AML
  • T-cell acute lymphoblastic leukemia T-ALL
  • diffuse large B-cell lymphoma testicular germ cell tumor
  • mesothelioma mesothelioma
  • esophagus carcinoma Merkel Cells cancer
  • MSI-high cancer KRAS-mutated tumors
  • adult T-cell leukemia/lymphoma and myelodys
  • basal cell carcinoma for example, selected from the group consisting of basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer , endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, gastrointestinal cancer, glioblastoma, liver cancer, hepatic neoplasia, intraepithelial neoplasia, kidney or kidney cancer, laryngeal cancer, liver cancer, lung cancer, Small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer , respiratory cancer, saliva
  • the aforementioned subject has a condition associated with PD-L1.
  • the subject's condition includes cancer that aberrantly expresses or does not aberrantly express PD-L1, and further includes non-metastatic or non-invasive and invasive or metastatic cancer.
  • methods of activating the immune system of a subject methods of activating cytotoxic T cells (CTL) in a subject in vivo or in vitro, methods of increasing proliferation of CD4 + and/or CD8 + T cells in vivo or in vitro Method, providing a method for activating NK cells in a subject in vivo or in vitro, providing a method for activating ⁇ T cells in a subject in vivo or in vitro, providing a method for activating Th1 cells in a subject in vivo or in vitro, providing activation, reduction or elimination
  • a method for the cell number and/or activity of at least one of regulatory T cells (Treg) in a subject providing a method for increasing IFN- ⁇ production and/or secretion of pro-inflammatory cytokines in a subject, Provide a method for blocking or inhibiting the interaction between PD-1 and PD-L1/PD-L2 in a subject, all of which include administering to the subject an effective amount of the aforementioned
  • the present disclosure provides detection applications of PD-1 antibodies or antigen-binding fragments thereof, immunoconjugates, polynucleotides, and compositions.
  • the present disclosure also provides a method, system or device for detecting PD-1 in vivo or in vitro, which includes treating the sample with the aforementioned binding protein, polynucleotide, and composition of the present disclosure.
  • an in vitro detection method, system or device may, for example, include:
  • a change eg, a statistically significant change in complex formation in a sample or subject, as compared to a control sample or subject, is indicative of the presence of PD-1 in the sample.
  • the in vivo detection method, system or device may comprise:
  • Detection can include determining where or when a complex is formed.
  • the aforementioned binding proteins and polynucleotides are labeled with detectable substances, and the detection of substances capable of binding proteins and polynucleotides (such as PD-1) is realized by detecting the labels.
  • Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances.
  • Complex formation of binding proteins, polynucleotides and PD-1 can be detected by measuring or visualizing what binds or does not bind to PD-1.
  • Conventional detection assays may be used, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or tissue immunohistochemistry.
  • binding proteins, polynucleotides of the disclosure can be labeled with fluorophore chromophores.
  • kits in some embodiments, and the kit includes the aforementioned binding protein, polynucleotide, and instructions for use in diagnosis.
  • the kit may also contain at least one additional reagent, such as a marker or an additional diagnostic agent.
  • additional reagent such as a marker or an additional diagnostic agent.
  • antibodies can be formulated as pharmaceutical compositions.
  • Figure 1A-1B is the result of detecting the activity of anti-PD-1 Nanobody by PD-1/PD-L1NFAT reporter gene system, using Pembrolizumab as a positive control, and human IgG4 as a negative control; among them, Figure 1A is A6_IgG4 and A17_IgG4 results, Figure 1B shows the results of A17h1_IgG4, A17m09_hIgG4, A17m0901_hIgG4, A17m0902_hIgG4, A17m0903_hIgG4, A17m0905_hIgG4.
  • Figure 2 is the detection of the degree of activation of T cells by A17m09_hIgG4 through the release of cytokines in the DC cell:T cell mixed lymphatic reaction, using pembrolizumab as a positive control and hIgG4 as a negative control.
  • Figure 3A and Figure 3B are the in vivo drug efficacy test results of A17m09_hIgG4 inhibiting the growth of mouse M38 colon cancer tumors. as a negative control.
  • Fig. 4 is the construction of the fusion protein of anti-PD-1 antibody and IL-2 mutant.
  • the anti-PD-1 Nanobody A17m0902 is coupled to the N-terminals of the Knob and Hole chains through the IgG1 heavy chain hinge region; in the molecular structure shown in Figure 4 B, the two The anti-PD-1 nanobody A17m0902 is connected end-to-end through the (G 4 S) 2 linker, and then its C-terminus is coupled to the N-terminus of the Knob and Hole chains through the IgG1 heavy chain hinge region;
  • Figure 4 C In the molecular structure, the anti-PD-1 nanobody A17m0902 is coupled to the N-terminal of the Hole chain through the IgG1 heavy chain hinge region, and the heavy chain of the Fab fragment corresponding to the anti-PD-1 antibody pembrolizumab is coupled to the IgG1 heavy chain hinge region.
  • the IL-2 mutant is connected to the C-terminus of the Knob chain of Fc through the (G 4 S) 3 linker, and in the molecule of F, IL-2v (V91T) is connected to the Knob chain through the G 4 S linker The N-terminal coupling.
  • Figure 5A and Figure 5B are the detection results of the proliferative activity of different forms of PD-1-IL-2v(V91T) fusion proteins on M07e and M07e-hPD-1 cells.
  • Figure 5A is the result of the proliferative activity of different forms of PD-1-IL-2v (V91T) fusion proteins on M07e-hPD-1 cells
  • Figure 5B is the results of different forms of PD-1-IL-2v (V91T) fusion proteins Results of the proliferative activity of PD-1-IL-2v(V91T) fusion protein on M07e cells.
  • Figure 6A and Figure 6B are the detection results of the proliferation activity of the same form of PD-1-IL-2v fusion protein with different IL-2 mutants on M07e and M07e-hPD-1 cell lines.
  • Figure 6A is the result of the proliferative activity of the PD-1-IL-2v fusion protein on M07e-hPD-1 cells
  • Figure 6B is the result of the proliferative activity of the PD-1-IL-2v fusion protein on M07e cells.
  • Fig. 7 is the test result of pro-proliferation activity of PD-1-IL-2v or IgG-IL-2v on M07e-hPD-1 cells.
  • Figure 8A and Figure 8B are the detection results of the proliferative activity of PD-1-IL-2v with different IL-2 mutants on unstimulated T cells. Among them, Fig. 8A is the result of CD4 + T cell proliferation, and Fig. 8B is the result of CD8 + T cell proliferation.
  • Figure 9A and Figure 9B are the detection results of the proliferative activity of PD-1-IL-2v with different IL-2 mutants on T cells stimulated by anti-CD3 antibody. Among them, Fig. 9A is the result of CD4 + T cell proliferation, and Fig. 9B is the result of CD8 + T cell proliferation.
  • Figure 10 shows the detection results of the proliferative activity of PD-1-IL-2v with different anti-PD-1 nanobody mutants on the M07e-hPD-1 cell line.
  • Figure 11 shows the tumor growth inhibition results of A-N88D on the mouse CT26 tumor model. Among them, A17m0902_hIgG4 was used as the control.
  • Fig. 12 shows the results of body weight changes in the mouse CT26 tumor model after administration of A-N88D.
  • the term “immunoconjugate” refers to a fusion polypeptide molecule comprising an effector moiety and at least one antigen binding moiety, optionally, the immunoconjugate may also comprise an Fc region.
  • the immunoconjugate comprises an effector moiety, an antigen binding moiety and an Fc region.
  • the immunoconjugate comprises an effector moiety, two antigen binding moieties and two Fc regions.
  • the immunoconjugate comprises one effector moiety, four antigen binding moieties and two Fc regions.
  • the immunoconjugate comprises an effector moiety, an antigen binding moiety and two Fc regions.
  • the immunoconjugate comprises one effector moiety, three antigen binding moieties and two Fc regions.
  • the immunoconjugate as described herein is a fusion protein, ie the components of the immunoconjugate are linked to each other by peptide bonds directly or by a linker.
  • the antigen binding moiety in the "immunoconjugate” is a PD-1 binding domain and the effector moiety is IL-2 or a variant thereof.
  • Programmed death 1 "Programmed death 1", “programmed cell death 1”, “protein PD-1”, “PD-1”, “PDCD1” and “hPD-1” are used interchangeably and include variants of human PD-1 Individuals, isotypes, species homologues, and analogs that share at least one common epitope with PD-1.
  • the complete PD-1 sequence can be found from GenBank accession number U64863.
  • “Programmed death ligand-1 (PD-L1)” is one of two cell surface glycoprotein ligands of PD-1 (the other being PD-L2), which downregulates T cells when bound to PD-1 Activation and cytokine secretion.
  • PD-L1 as used herein includes human PD-L1 (hPD-L1), variants, isoforms, and interspecies homologs of hPD-L1, and hPD-L1 having at least one common epitope analogues of .
  • the complete hPD-L1 sequence can be found with GenBank accession number Q9NZQ7.
  • PD-1 binding domain or "PD-1 binding protein” are used interchangeably and mean any protein capable of specifically binding PD-1 or any molecule comprising said protein.
  • a PD-1 binding protein may comprise an antibody against PD-1 as defined in the present disclosure, an antigen-binding fragment thereof or a conjugate thereof.
  • PD-1 binding proteins also encompass immunoglobulin superfamily antibodies (IgSF) or CDR grafted molecules.
  • the "PD-1 binding protein” of the present disclosure may comprise at least one immunoglobulin single variable domain (such as VHH) that binds to PD-1.
  • a "PD-1 binding protein” may comprise 2, 3, 4 or more immunoglobulin single variable domains (eg, VHH) that bind PD-1.
  • the PD-1 binding protein of the present disclosure may also contain linkers and/or moieties with effector functions, such as half-life prolonging moieties (such as immunoglobulin binding to serum albumin). globulin single variable domain), and/or a fusion partner (such as serum albumin) and/or a conjugated polymer (such as PEG) and/or an Fc region.
  • linkers and/or moieties with effector functions such as half-life prolonging moieties (such as immunoglobulin binding to serum albumin).
  • globulin single variable domain and/or a fusion partner (such as serum albumin) and/or a conjugated polymer (such as PEG) and/or an Fc region.
  • a "PD-1 binding protein" of the present disclosure also encompasses bi/multispecific antibodies comprising immunoglobulins that bind different antigens (such as a primary antibody that binds a first antigen (such as PD-1) And a second antibody that binds to a second antigen (such as 4-1BB), optionally includes a third antibody that binds to a third antigen, and further optionally includes a fourth antibody that binds to a fourth antigen.
  • a "PD-1 binding protein” or “PD-1 antibody or antigen-binding fragment thereof” of the present disclosure may, for example, comprise one or more effector molecules in a conjugated manner.
  • effector molecules include, for example, antineoplastic agents, drugs, toxins, biologically active proteins (such as enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof (such as DNA, RNA and fragments thereof ), radionuclides, especially radioiodides, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds detectable by NMR or ESR spectroscopic analysis.
  • the effector molecule is a cytokine (eg, IL-2, IL-15, or variants thereof).
  • the effector molecule is a polymer, which may generally be a synthetic or naturally occurring polymer, specific examples of synthetic polymers include optionally substituted linear or branched poly(ethylene glycol), poly( propylene glycol), poly(vinyl alcohol) or derivatives thereof, specific naturally occurring polymers include lactose, amylose, dextran, glycogen or derivatives thereof.
  • the polymer is albumin or a fragment thereof, such as human serum albumin or a fragment thereof. The conjugation of the polymer to the PD-1 binding protein or PD-1 antibody can be achieved by conventional methods.
  • Cytokines are the general term for proteins released by a population of cells that act as intercellular mediators on other cells. Examples of such cytokines include lymphokines, monokines, chemokines and traditional polypeptide hormones. Exemplary cytokines include: human IL-2, IL-15, IFN- ⁇ , IL-6, TNF ⁇ , IL-17 and IL-5 and variants thereof.
  • Interleukin-2 refers to any native IL-2 from any vertebrate source, including mammals such as primates (eg, humans) and rodents (eg, mice and rats).
  • the term encompasses unprocessed IL-2 as well as any form of IL-2 that results from processing in the cell.
  • the term also encompasses naturally occurring variants of IL-2, such as splice variants or allelic variants.
  • the amino acid sequence of an exemplary wild-type human IL-2 is shown in SEQ ID NO: 87.
  • Unprocessed human IL-2 additionally includes a signal peptide of N-terminal 20 amino acids (as shown in SEQ ID NO.272 in WO2012107417), which is lacking in mature IL-2 molecules.
  • “Mutated IL-2” or “IL-2 variant” refers to a polypeptide that differs from the amino acid sequence of wild-type IL-2 due to a mutation (eg, substitution, deletion, addition) at at least one amino acid position.
  • IL-2 variants single amino acid substitutions are described in the following manner: [original amino acid residue/position/substituted amino acid residue].
  • F42A a substitution of phenylalanine at position 42 for alanine
  • Single amino acid substitutions can be linked by "/" to indicate combined mutations at multiple given positions.
  • a combined mutation at the position of F42A, L72G and N88R can be expressed as: F42A/L72G/N88R, which means that the variant contains F42A, L72G and N88R mutations.
  • amino acid positions in the IL-2 protein or IL-2 sequence segment when referring to the amino acid position in the IL-2 protein or IL-2 sequence segment, by referring to the amino acid sequence SEQ ID NO:87 of the wild-type human IL-2 protein (also called wt IL-2) make sure.
  • amino acid positions on other IL-2 proteins or polypeptides can be identified by amino acid sequence alignment with SEQ ID NO:87. Therefore, in this disclosure, unless otherwise stated, the amino acid positions of the IL-2 protein or polypeptide are the amino acid positions numbered according to SEQ ID NO:87.
  • F42 refers to the 42nd phenylalanine residue F of SEQ ID NO:40, or the amino acid residue at the corresponding position on other IL-2 polypeptide sequences after alignment. Sequence alignments for amino acid position determination can be performed using the Basic Local Alignment Search Tool available at https://blast.ncbi.nlm.nih.gov/Blast.cgi with default parameters.
  • CD25 or "alpha subunit of IL-2 receptor” refers to any native CD25 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g. mice and rats), including "Full-length” unprocessed CD25, as well as any form of CD25 derived from processing in a cell, also includes naturally occurring variants of CD25, such as splice variants or allelic variants. In certain embodiments, CD25 is human CD25.
  • High affinity IL-2 receptor refers to the heterotrimeric form of the IL-2 receptor, which consists of the receptor gamma subunit (also known as the general cytokine receptor gamma subunit, gammac, or CD132), the receptor ⁇ subunit (also known as CD122 or p70) and receptor ⁇ subunit (also known as CD25 or p55).
  • an "intermediate affinity IL-2 receptor” refers to an IL-2 receptor comprising only a gamma subunit and a beta subunit without an alpha subunit (see e.g. Olejniczak and Kasprzak, MedSci Monit 14, RA179-189 (2008 )).
  • Antibody is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal Clonal antibodies; monospecific antibodies, multispecific antibodies (eg, bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), so long as they exhibit the desired antigen-binding activity.
  • Antibody can refer to immunoglobulin, which is a tetrapeptide chain structure formed by two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , ⁇ chain and ⁇ chain.
  • the same class of Ig can be divided into different subclasses according to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either kappa chains or lambda chains by difference in the constant region.
  • Each of the five Ig classes can have either a kappa chain or a lambda chain.
  • the sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region (C region).
  • the variable region includes 3 hypervariable regions (CDRs) and 4 framework regions (FRs) with relatively conserved sequences. Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDR).
  • Each light chain variable region (VL) and heavy chain variable region (VH) are composed of 3 CDR regions and 4 FR regions, and the sequence from the amino terminal to the carboxyl terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2 and HCDR3.
  • Antigen-binding fragment encompasses single chain antibodies (i.e. full length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv , single domain antibody (e.g. VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibody, Bis-scFv, diabody, tribody, triabody, tetrabody and epitope binding fragments of any of the above (see e.g. Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217).
  • the antigen-binding fragment is VHH.
  • Methods for generating and preparing these antigen-binding fragments are well known in the art (see eg Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).
  • the Fab-Fv format was first disclosed in WO2009/040562, and its disulfide bond-stabilized form Fab-dsFv was first disclosed in WO2010/035012.
  • Antigen-binding fragments of the present disclosure also include Fab and Fab' fragments described in WO2005/003169, WO2005/003170 and WO2005/003171.
  • Multivalent antibodies may comprise multiple specificities such as bispecifics or may be monospecific (see e.g. WO92/22583 and WO05/113605), an example of the latter being the Tri-Fab (or TFM).
  • Fc domain or “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. It includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain can vary slightly, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or Pro230 to the carboxy-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage, whereby one or more, especially one or two amino acids are excised from the C-terminus of the heavy chain.
  • an antibody produced by a host cell by expressing a specific nucleic acid molecule encoding a full-length heavy chain may include a full-length heavy chain, or it may include a cleavage variant of a full-length heavy chain (also referred to in this disclosure as a "cleavage variant heavy chain”). chain”).
  • a cleavage variant heavy chain also referred to in this disclosure as a "cleavage variant heavy chain”
  • chain When the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index) may be the case.
  • the C-terminal lysine (K447), or the C-terminal glycine (G446) and lysine (K447) of the Fc region may or may not be present.
  • the amino acid sequence of the heavy chain comprising the Fc region is expressed in the present disclosure without the C-terminal glycine-lysine dipeptide.
  • the heavy chain of one subunit of the Fc region comprised in the immunoconjugate comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to Kabat's EU index).
  • the heavy chain of one subunit of the Fc region comprised in the immunoconjugate comprises an additional C-terminal glycine residue (G446, numbering according to Kabat's EU index).
  • a composition (eg, a pharmaceutical composition) of the disclosure comprising a population of immunoconjugates of the disclosure.
  • the population of immunoconjugates can comprise molecules with full-length heavy chains and molecules with cleaved variant heavy chains.
  • the population of immunoconjugates may consist of a mixture of molecules with full-length heavy chains and molecules with cleaved variant heavy chains, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the immunoconjugates
  • the compound has a cleavage variant heavy chain that can be the C-terminal lysine (K447) of the Fc region, or the C-terminal glycine (G446) and lysine (K447) are absent.
  • the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • a “subunit” of an Fc region refers to one of the two polypeptides that form a dimeric Fc region, ie, a polypeptide comprising a C-terminal constant region in an immunoglobulin heavy chain capable of stabilizing its association.
  • subunits of the IgG Fc region comprise IgG CH2 and IgG CH3 constant domains.
  • first subunit or Fc1 “second subunit” or Fc2 is not intended to confer a specific order or orientation, but is used for ease of distinction.
  • a "modification (or mutation) that promotes the association of the first and second subunits of an Fc domain” is a manipulation of the peptide backbone or Fc domain that reduces or prevents the association of a polypeptide comprising a subunit of the Fc domain with the same polypeptide to form a homodimer.
  • Post-translational modification of domain subunits include separate modifications to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) for which association is desired, wherein The modifications are complementary to each other, thereby facilitating the association of the two Fc domain subunits.
  • association-promoting modifications may alter the structure or charge of one or both Fc domain subunits to facilitate their association sterically or electrostatically, respectively.
  • (hetero)dimerization occurs between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit in the presence of additional components fused to each subunit (e.g. an antigen binding moiety). ) may be different in the sense that they are different.
  • the association-promoting modification comprises amino acid mutations, specifically amino acid substitutions, in the Fc domain.
  • “Complementarity” refers to the combination of interactions affecting heavy chain/light chain pairing, eg, at the interface of CH1 and CL (or CH3 and CH3) of the antigen binding proteins described herein.
  • “Steric complementarity” or “conformational complementarity” refers to the compatibility of three-dimensional structures at the interacting surfaces of, for example, CH1 and CL (or CH3 and CH3).
  • “Electrostatic complementarity” refers to the compatibility of placing negatively and/or positively charged atoms at the interacting surfaces of eg CH1 and CL (or CH3 and CH3). Methods of measuring electrostatic complementarity at protein/protein interfaces are known in the art and described, for example, in McCoy et al.
  • ADCC antibody effector functions in reference to antibody use refer to those biological activities attributable to the Fc region of an antibody that vary with the antibody isotype.
  • Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor (FcR) binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) , cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (eg, B-cell receptors), and B-cell activation.
  • ADCC is an immune mechanism leading to lysis of antibody-coated target cells by immune effector cells.
  • a target cell is a cell to which an antibody comprising an Fc region or a derivative thereof typically binds specifically via the protein portion at the N-terminus of the Fc region.
  • "Reduced ADCC” is defined as the reduction in the number of target cells lysed within a given time period with a given concentration of antibody in the medium surrounding the target cells by the ADCC mechanism defined above, and/or achieved by the ADCC mechanism for a given The increase in antibody concentration in the medium surrounding the target cells required to lyse a given number of target cells within a given period of time.
  • the reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cell but not yet engineered using the same standard production, purification, formulation and storage methods known to those skilled in the art.
  • ADCC mediated by an antibody comprising an ADCC-reducing amino acid substitution in its Fc domain is relative to ADCC mediated by the same antibody without such an amino acid substitution in the Fc domain.
  • Suitable assays for measuring ADCC are well known in the art (see eg WO2006/082515 or WO2012/130831).
  • activating Fc receptor is an Fc receptor that, upon engagement of the Fc domain of an antibody, initiates signaling events that stimulate cells bearing the receptor to perform effector functions.
  • Human activating Fc receptors include FcyRIIIa (CD16a), FcyRI (CD64), FcyRIIa (CD32) and FcaRI (CD89).
  • Immunoconjugate refers to a polypeptide molecule that includes at least one IL-2 molecule and at least one antibody. As described herein, IL-2 molecules can be linked to antibodies through a variety of interactions and in a variety of configurations. In specific embodiments, the IL-2 molecule is fused to the antibody via a peptide linker. Certain immunoconjugates according to the invention consist essentially of one IL-2 molecule and antibody linked by one or more linker sequences.
  • Reduced avidity or “reduced binding”, eg decreased binding to IL-2R ⁇ , refers to a decreased affinity for the corresponding interaction, eg as measured by SPR. The term also includes the reduction of the affinity to zero or below the detection limit of the analytical method, ie the complete elimination of the interaction. Conversely, “reduced affinity” or “increased binding” refers to an increase in the binding affinity of the corresponding interaction.
  • deterministic delineation of the CDRs and identification of the residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and/or resolving the structure of the antibody-ligand complex. This can be achieved by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography.
  • Various analytical methods can be used to identify CDRs, including but not limited to Kabat numbering system, Chothia numbering system, AbM numbering system, IMGT numbering system, contact definition, conformation definition.
  • the Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see eg Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8).
  • the Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the location of certain structural ring regions. (See eg Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83).
  • the AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group for modeling antibody structures (see, e.g., Martin et al., 1989, ProcNatl Acad Sci (USA), 86:9268-9272; "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd).
  • the AbM numbering system uses a combination of knowledge base and ab initio methods to model the tertiary structure of antibodies from basic sequences (see Samudrala et al., 1999, "AbM” in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198. Initio Protein Structure Prediction Using a Combined Hierarchical Approach” described).
  • a CDR may refer to a CDR defined by any method known in the art, including combinations of methods. The correspondence between various numbering systems is well known to those skilled in the art, and is exemplary, as shown in Table 1 below.
  • the CDR amino acid residues of the VL and VH regions of the disclosed antibodies conform to the known Kabat numbering system in number and position.
  • Antibodies of the present disclosure may be polyclonal, monoclonal, xenogeneic, allogeneic, isogenic, or modified forms thereof, with monoclonal antibodies being particularly useful in various embodiments.
  • antibodies of the present disclosure are recombinant antibodies.
  • “recombinant” generally refers to products such as cells or nucleic acids, proteins or vectors, and means that the cells, nucleic acids, proteins or vectors have been modified by introducing heterologous nucleic acids or proteins or changing natural nucleic acids or proteins, or that the Said cells are derived from cells so modified.
  • a recombinant cell expresses a gene that is not present in the native (non-recombinant) form of the cell or expresses a native gene that would otherwise be aberrantly expressed, underexpressed, or not expressed at all.
  • a “domain” of a polypeptide or protein refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein.
  • a domain is responsible for a single functional property of a protein, and in many cases can be added, removed or transferred to other proteins without loss of the rest of the protein and/or the function of the domain.
  • Immunoglobulin domain refers to a globular region of an antibody chain (eg, a chain of a conventional tetrapeptide chain antibody or a chain of a heavy chain antibody), or a polypeptide consisting essentially of such a globular region. Immunoglobulin domains are characterized by their maintenance of the immunoglobulin fold characteristic of antibody molecules, consisting of a 2-layer sandwich of approximately seven antiparallel beta-sheet strands, optionally stabilized by conserved disulfide bonds, arranged in two beta-sheets .
  • Immunoglobulin variable domain refers to the term “framework region 1" or “FR1”, “framework region 2” or “FR2”, “framework region 3” or “FR3” essentially defined in the art and hereinafter as “FR1” or “FR1”, respectively. , and four “framework regions” of “framework region 4" or “FR4", wherein the framework regions are respectively referred to in the art and hereinafter as “complementarity determining region 1" or "CDR1", The three “complementarity determining regions” or “CDRs” of “complementarity determining region 2" or “CDR2”, and “complementarity determining region 3" or “CDR3” are spaced apart.
  • an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains are endowed with antigen-specificity by having an antigen-binding site.
  • Immunoglobulin single variable domain is commonly used to refer to a domain that can be used without interaction with other variable domains (e.g., without the need between the VH and VL domains of a conventional four-chain monoclonal antibody).
  • an immunoglobulin variable domain (which may be a heavy or light chain domain, including a VH, VHH or VL domain) that forms a functional antigen binding site.
  • immunoglobulin single variable domains include Nanobodies (including VHH, humanized VHH and/or camelized VH, such as camelized human VH), IgNAR, domains, as VH domains or derived from VH Domain (single domain) antibodies (eg dAbsTM) and (single domain) antibodies that are or are derived from VL domains (eg dAbsTM).
  • Immunoglobulin single variable domains based on and/or derived from heavy chain variable domains are generally preferred.
  • a specific example of an immunoglobulin single variable domain is a "VHH domain” (or simply "VHH") as defined below.
  • VHH domains also known as heavy chain single domain antibodies, VHH, VHH antibody fragments, VHH antibodies, nanobodies, are antigen-binding immunoglobulins known as “heavy chain antibodies” (ie, “antibodies lacking light chains") Protein variable domains (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature363, 446-448( 1993)).
  • VHH domain is used to distinguish the variable domain from the heavy chain variable domain (which is referred to as a "VH domain” in this disclosure) and the light chain variable domain present in conventional tetrapeptide chain structured antibodies. domain (which is referred to as a "VL domain” in this disclosure).
  • the VHH domain specifically binds the epitope without the need for additional antigen-binding domains (in contrast to the VH or VL domains in conventional tetrapeptide chain antibodies, in which case the epitope is recognized by the VL domain together with the VH domain) .
  • the VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain.
  • VHH domain includes but is not limited to natural antibodies produced by camelids, and can also be antibodies produced by camelids After humanization, it can also be obtained by screening by phage display technology.
  • the VHH domain comprises three CDRs and four framework regions, designated as FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • the VHH domain can be truncated at the N-terminus or C-terminus so that it only comprises part of FR1 and/or FR4, or lacks one or both of those framework regions, as long as the VHH remains substantially Antigen binding and specificity will suffice.
  • the total number of amino acid residues in the VHH domain will generally range from 110 to 120, often between 112 and 115. Note however that smaller and longer sequences may also be suitable for this invention. Disclose the stated purpose.
  • the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e. one according to Kabat numbering or positions may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering).
  • Kabat numbering i.e. one according to Kabat numbering or positions may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering.
  • numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.
  • Other numbering systems or coding conventions include Chothia, IMGT, AbM.
  • Humanized antibody also known as CDR-grafted antibody (CDR-grafted antibody) refers to an antibody produced by grafting a non-human CDR sequence into a human antibody variable region framework. It can overcome the strong immune response induced by chimeric antibodies due to carrying a large number of non-human protein components. In order to avoid a decrease in activity while reducing immunogenicity, minimal reverse mutations can be performed on the variable region of the fully human antibody to maintain activity.
  • Examples of "humanization” include that the VHH domain derived from Camelidae can be replaced by the amino acid sequence of the original VHH sequence with one or more amino acid residues present in the corresponding position in the VH domain of an antibody with a conventional tetrapeptide chain structure.
  • Humanization by one or more amino acid residues in VHH (also referred to as “sequence optimization” in this disclosure. Mutations are other modifications to the sequence, such as removal of potential post-translational modification sites).
  • a humanized VHH domain may contain one or more fully human framework region sequences, and in some embodiments, may contain the human framework region sequences of IGHV3.
  • Humanization methods such as protein surface amino acid humanization (resurfacing) and antibody humanization universal framework grafting method (CDR grafting to a universal framework), that is, CDR “grafting” to other "scaffolds" (including but not limited to human scaffolds or non-immunoglobulin scaffold). Scaffolds and techniques suitable for such CDR grafting are known in the art.
  • the germline DNA sequences of the human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database, and in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition.
  • the humanized antibodies of the present disclosure also include humanized antibodies that are further subjected to affinity maturation of CDRs by phage display.
  • minimal reverse mutations or back mutations can be performed on the human antibody variable region framework sequence to maintain activity.
  • an “affinity matured” antibody is one that possesses one or more alterations in one or more hypervariable regions (HVRs) that result in the antibody's ability to respond to the antigen as compared to a parental antibody that does not possess such alterations. affinity improve.
  • HVRs hypervariable regions
  • an "avidity matured" PD-1 antibody has one or more changes in one or more CDRs that result in an increased affinity for the antigen compared to its parental antibody.
  • Affinity matured antibodies can be prepared by methods known in the art, for example, from Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc. Nat. Acad.
  • the PD-1 antibodies, immunoconjugates of the present disclosure will be present at preferably 10 ⁇ 7 to 10 ⁇ 10 moles/liter (M), more preferably 10 ⁇ 8 to 10 ⁇ as measured in a Biacore or KinExA or Fortibio assay. 10 mol/L, even more preferably 10 -9 to 10 -10 or lower dissociation constant (KD), and/or at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M , more preferably an association constant (KA) of at least 10 ⁇ 10 M for binding to the antigen to be bound (ie PD-1). Any KD value greater than 10 -4 M is generally considered to indicate non-specific binding.
  • Specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and/or competitive binding assays as described in this disclosure (for example radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competitive assay).
  • SPR surface plasmon resonance
  • RIA radioimmunoassay
  • EIA enzyme immunoassay
  • sandwich competitive assay sandwich competitive assay
  • Antigen refers to a molecule used to immunize an immunocompetent vertebrate, to generate antibodies that recognize the antigen, or to screen expression libraries (such as, inter alia, phage, yeast or ribosome display libraries).
  • antigen is defined more broadly to include target molecules specifically recognized by antibodies, and to include portions or mimetics of molecules used in immunization procedures for antibody generation or library screening for antibody selection.
  • monomers and multimers such as dimers, trimers, etc.
  • truncated variants of human PD-1 and other The variants are all called PD-1 antigens.
  • Epitope refers to the site on an antigen to which an immunoglobulin or antibody binds. Epitopes can be formed from contiguous amino acids, or non-contiguous amino acids that are juxtaposed by the tertiary folding of the protein. Epitopes formed from adjacent amino acids are generally maintained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally lost upon denaturing solvent treatment.
  • An epitope typically comprises at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation assays, among others. Methods of determining the spatial conformation of an epitope include techniques in the art and techniques described in this disclosure, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.
  • Specific binding and “selective binding” refer to the binding of an antibody to a predetermined epitope on an antigen.
  • an antibody when used as an analyte and an antibody is used as a ligand, when the surface plasmon resonance (SPR) technique is used in an instrument, the antibody is approximately below 10 ⁇ 7 M or even smaller
  • the equilibrium dissociation constant (K D ) binds to the predetermined antigen or its epitope, and its binding affinity to the predetermined antigen or its epitope is higher than that of the predetermined antigen (or its epitope) or closely related antigens At least twice the binding affinity of non-specific antigens (such as BSA, etc.).
  • An "antibody that recognizes an antigen” may be used interchangeably with a “specifically binding antibody” in this disclosure.
  • Binding affinity or “avidity” is used in the present disclosure as a measure of the strength of a non-covalent interaction between two molecules (eg, an antibody or portion thereof and an antigen).
  • the binding affinity between two molecules can be quantified by determining the dissociation constant (KD).
  • KD can be determined by measuring the kinetics of complex formation and dissociation using, for example, the Surface Plasmon Resonance (SPR) method (Biacore).
  • SPR Surface Plasmon Resonance
  • the rate constants corresponding to the association and dissociation of the monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively.
  • the value of the dissociation constant can be determined directly by well known methods and can be calculated even for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9:340-362).
  • KD can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432).
  • Other standard assays to assess the binding ability of antibodies against a target antigen are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays exemplified elsewhere in this disclosure.
  • Binding kinetics and binding affinity of antibodies can also be assessed by standard assays known in the art, such as surface plasmon resonance (SPR), for example by using the Biacore TM system or KinExA. Binding affinities associated with different molecular interactions can be compared by comparing the KD values of the individual antibody/antigen complexes, eg, a comparison of the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the KD value of an interaction of interest (e.g., a specific interaction between an antibody and an antigen) with that of an unintended interaction (e.g., a control antibody known not to bind CD40). KD value was evaluated.
  • SPR surface plasmon resonance
  • a “conservative substitution” refers to a substitution for another amino acid residue that has similar properties to the original amino acid residue.
  • lysine, arginine, and histidine have similar properties in that they have basic side chains
  • aspartic acid and glutamic acid have similar properties in that they have acidic side chains.
  • glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains
  • alanine , valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have nonpolar side chains.
  • “Homology”, “identity” or “sequence identity” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in two compared sequences is occupied by the same nucleotide or amino acid monomer, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position of.
  • the percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of compared positions x 100%. For example, two sequences are 60% homologous if there are 6 matches or homology at 10 positions in the two sequences when the sequences are optimally aligned. In general, comparisons are made when two sequences are aligned to yield the greatest percent homology.
  • Nucleic acid molecule “nucleic acid” or “polynucleotide” are used interchangeably to refer to DNA and RNA. Nucleic acid molecules may be single- or double-stranded, preferably double-stranded DNA. A nucleic acid is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the coding sequence transcription of the sequence, a promoter or enhancer is operably linked to the coding sequence.
  • Vector means a construct capable of delivering, and in some embodiments expressing, one or more genes or sequences of interest in a host cell.
  • vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors conjugated to cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes , and certain eukaryotic cells such as producer cells.
  • a “host cell” includes any cell or cell culture that can be or has been a recipient of a vector for the incorporation of a polynucleotide insert.
  • a host cell includes the progeny of a single host cell, and the progeny may not necessarily be identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental or deliberate mutation.
  • Host cells include cells transfected and/or transformed in vivo with polynucleotides of the present disclosure.
  • Cell “cell line,” and “cell culture” are used interchangeably, and all such designations include progeny. It should also be understood that all progeny may not be precisely identical in DNA content due to deliberate or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cells are included.
  • “Pharmaceutical composition” means a mixture containing one or more of the antibodies described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, and other components such as a physiologically/pharmaceutically acceptable carrier and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thus exert biological activity.
  • a “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate buffered saline, water, emulsions such as oil/water emulsions, and various types of wetting agents.
  • the diluent for aerosol or parenteral administration is phosphate buffered saline (PBS) or physiological (0.9%) saline.
  • Compositions comprising such carriers are formulated by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18th ed., A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and R Remington, The Science and Practice of Pharmacy 20th ed. Mack Publishing, 2000).
  • Inhibition or “blocking” are used interchangeably and encompass both partial and complete inhibition/blocking. “Inhibition of growth” (eg, in relation to cells) is intended to include any measurable decrease in cell growth.
  • Proliferative disease refers to a disorder associated with some degree of abnormal cell proliferation.
  • the proliferative disorder is cancer.
  • Cancer refers to or describes a physiological disorder in mammals that is often characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
  • cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal carcinoma , hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, liver tumor, breast cancer, colon cancer, Cancer of the rectum, colorectum, endometrium or uterus, salivary gland, kidney, prostate, vulva, thyroid, liver, anus, penis Carcinoma, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-
  • cancers suitable for treatment by PD-1 binding proteins of the present disclosure include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin's lymphoma NHL, renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple Myeloma.
  • the cancer is selected from the group consisting of: non-small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer (e.g.
  • the cancer is selected from the group consisting of non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer (eg, triple negative breast cancer), including metastatic forms of those cancers.
  • Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • Preventing cancer means delaying, inhibiting or preventing the onset of cancer in a subject in which the onset of cancer or tumorigenesis has not been proven, but has been determined, for example, by genetic screening or other methods, identified susceptibility to cancer. This also includes treating a subject with a precancerous condition to halt progression of the precancerous condition to a malignancy or to cause regression thereof.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid refers to the interaction of an exogenous drug, therapeutic, diagnostic, or composition with an animal. , humans, subjects, cells, tissues, organs or biological fluids, such as therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of cells includes contacting the reagents with the cells, and contacting the reagents with a fluid, wherein the fluid contacts the cells.
  • administering also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnostic, binding composition or by another cell. When applied to human, veterinary or research subjects, it refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent to a subject, for example, comprising any antibody of the present disclosure or a pharmaceutical composition thereof as a therapeutic agent, the subject has suffered from, is suspected of having, or tends to suffer from There is one or more proliferative diseases or symptoms thereof for which the therapeutic agent is known to have a therapeutic effect.
  • a therapeutic agent is administered in a subject or population to be treated in an amount effective to alleviate one or more symptoms of a disease, either by inducing regression of such symptoms or inhibiting the progression of such symptoms to any clinically measurable extent.
  • the amount of a therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on factors such as the disease state, age, and weight of the subject, and the extent to which the drug produces the desired therapeutic effect in the subject. ability. Any medication normally used by a physician or other professional health care professional to evaluate the severity or progression of symptoms Any clinical test method can evaluate whether the symptoms of the disease have been alleviated.
  • any statistical test method known in the art such as Student t-test, chi-square test, according to Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test determined that it should reduce the target disease symptoms in a statistically significant number of subjects.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to permit or facilitate diagnosis.
  • the effective amount for a subject may vary depending on factors such as the condition being treated, the general health of the subject, the method, route and dose of administration, and the severity of side effects.
  • An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • a subject of the present disclosure can be an animal or a human subject.
  • first”, “second”, and “third” is not intended to confer a specific order or orientation, but is used for ease of distinction, for example, when there is more than one module of the same type.
  • “/” when used for a mutation, it means that the mutations exist simultaneously in the same IL-2 variant or the same Fc or the same immunoconjugate, for example, "F42A/L72G” means in Both F42A and L72A mutations are present in the same IL-2 variant.
  • subject and patient mean mammals, especially primates, especially humans.
  • the numbering of amino acid positions of IL-2 or its variants in the present disclosure is relative to wild-type IL-2 (such as shown in SEQ ID NO: 87).
  • This disclosure uses human PD-1 with His tag as an immunization antigen, and human PD-1 with biotin PD-1 and cynomolgus monkey PD-1 were used as screening antigens, all of which were purchased from AcroBiosystems.
  • the human PD-1 protein (NP_005009.2) sequence is as follows, where the underline is the sequence of the extracellular region, and the amino acids start and end at Leu25-Gln 167.
  • Vaccine 2 healthy alpacas.
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • PBMCs in peripheral blood were isolated, RNA in PBMCs was extracted, and reverse transcription was performed to obtain total cDNA.
  • the cDNA was used as the template for the first round of nested PCR.
  • the second round of nested PCR product fragments were ligated with a yeast library vector (yeast library vector pYDN3), and the ligated products were transformed into yeast competent cells by electric shock.
  • the insertion rate and library diversity were verified by colony PCR. According to the number of library transformants, library insertion rate and library diversity analysis and sequencing results, the library capacity of one alpaca library was 9.28 ⁇ 10 7 , and that of the other alpaca library was 1.54 ⁇ 10 8 .
  • flow cytometry sorting For this yeast display system, flow cytometry (FACS) sorting, and library screening and identification are used for antibody screening.
  • the sorting adopts two rounds of sorting steps: the first round of sorting uses biotin-labeled human PD-1 protein (Avitag-His Tag) magnetic beads for enrichment, and the second round uses biotin-labeled monkey PD-1 protein (His, Avitag-His Tag) ) flow cytometry sorter (flow cytometry antibodies: mouse anti-HA tag Dylight 488 (Mouse anti-HA tag Dylight 488); streptavidin Dylight 650 (streptavidin Dylight 650)).
  • flow cytometry antibodies mouse anti-HA tag Dylight 488 (Mouse anti-HA tag Dylight 488); streptavidin Dylight 650 (streptavidin Dylight 650)
  • 18 unique sequences of VHH cross-linked with human and monkey PD-1 antigens were obtained. The sequences of A6 and A17 among them are
  • the above sequences were linked to human IgG4 Fc (including the hinge region, and with S228P, according to the EU numbering system) fragments to construct VHH-Fc antibodies.
  • the human IgG4 Fc (including the hinge region) sequence is as follows:
  • Pembrolizumab (purchased from Baiying Biotechnology) was used as a control, and the results are shown in Table 5. Moreover, the EC 50 value of A17_hIgG4 was 0.3568nM, which was significantly better than other antibodies obtained by simultaneous screening (results not shown).
  • Antibody humanization is carried out by CDR-grafting method.
  • the human germline gene IGHV 3-23 IGHV3-23*01; IGHV3-23*04
  • the CDR was transplanted to retain the FR2 of the nanobody 4 key residues (Y37, E44, R45, L47).
  • the CDR was transplanted to retain the FR2 of the nanobody 4 key residues (Y37, E44, R45, L47).
  • Humanized antibodies A17h1, A17h2, A17h3, A6h1 were obtained.
  • TCE removal modification was carried out.
  • antibody deamidation and antibody isomerization sites are modified. Get the following sequence:
  • A17m09 was further selected for germline transformation of antibody sequences to improve the degree of humanization, and the following sequences were obtained:
  • the CDR region of A17 of the present disclosure has the following general formula sequence:
  • CDR1 DYSMS (SEQ ID NO: 7)
  • CDR2 IISGSGX 1 X 2 X 3 HYVDSVKG, wherein X 1 is selected from V or G, X 2 is selected from I or S, X 3 is selected from T or A (SEQ ID NO: 36)
  • CDR3 VSDWX 4 X 5 Y, wherein, X 4 is selected from D or E, X 5 is selected from D or E (SEQ ID NO: 37)
  • CDR2 can be:
  • CDR3 can be:
  • VSDWDDY (SEQ ID NO: 9)
  • VSDWEDY (SEQ ID NO: 41)
  • Example 4 PD-1/PD-L1 reporter gene system detects the blocking of PD-1 by anti-PD-1 nanobody
  • the biological activity function of PD-1 antibody at the cell level can be detected.
  • the system consists of two genetically engineered cell lines: PD-1-overexpressing effector cell Jurkat (containing NFAT reporter gene luciferase) and PD-L1-overexpressing antigen-presenting cell CHO-K1 cells. When these two cells were co-cultured, the PD-1/PD-L1 interaction inhibited TCR-mediated NFAT activation, which in turn suppressed the expression of the NFAT reporter gene luciferase.
  • TCR activation induces the expression of luciferase through the NFAT pathway, which can be measured by adding Bio-Glo reagents and using a luminometer for fluorescence quantitative detection to determine the degree of activation of effector cells and Antibody biological activity.
  • Dilute CHO-K1 cells overexpressing PD-L1 to 4 ⁇ 10 5 /mL add 100 ⁇ L to each well of a 96-well plate, and culture overnight. After aspirating the medium, quickly add 40 ⁇ L of 1.25 ⁇ 10 6 /mL PD-1 Jurkat cells and 40 ⁇ L of anti-PD-1 nanobody solutions of different concentrations (diluted in 1640+2% FBS solution). Incubate at 37°C for 6 hours, return to room temperature, add 40 ⁇ L Bright-glo reagent (Cat: E2620, promega) to each well, shake at 350 rpm for 5 minutes in the dark, keep at room temperature for 5 minutes, and read the fluorescence value with a microplate reader. Calculation of IC50 .
  • Bright-glo reagent Cat: E2620, promega
  • Pembrolizumab and hIgG4 isotype were used as controls.
  • the biological function activity IC50 value of A17_hIgG4 is 1.199nM, which is significantly better than other antibodies screened simultaneously in the present disclosure (for example, A3_hIgG4 is 7.62, A13_hIgG4 is 3.208; A3_hIgG4 and A13_hIgG4 do not show the sequence) .
  • the anti-PD-1 nanobody was functionally verified, see Figure 1B and Table 7, the IC 50 activity of A17h1_hIgG4 was similar to that of the positive antibody pembrolizumab.
  • the biological activity IC 50 values of A17m0901_hIgG4 and A17m0902_hIgG4 antibodies were 0.5307nM and 0.4352nM, respectively, which were better than pembrolizumab.
  • PD-1/PD-L1NFAT reporter gene system detects the activity IC 50 of the modified anti-PD-1 nanobody
  • CM5 sensor chip was used in the experiment, and HBS-EP+buffer solution (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20) was used as the mobile phase.
  • HBS-EP+buffer solution (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20) was used as the mobile phase.
  • the anti-human IgG (Fc) antibody was prepared into a 30 ⁇ g/mL solution with 10 mM sodium acetate buffer (pH 5.0), and the Immobilization program was selected to automatically carry out amino-coupling immobilization of the anti-human IgG (Fc) antibody channel.
  • Each antibody to be tested was prepared as a ligand with HBS-EP+buffer solution, and captured with an anti-human IgG (Fc) antibody on the chip channel.
  • Human or cynomolgus PD-1 antigen protein (Sino Biological, 10377-H08H; 90311-C08H) was used as the analyte, prepared with HBS-EP+ buffer solution, and the analyte was serially diluted 2 times. Flow through the experimental channel and reference channel at a certain flow rate, combine for 1 min, and dissociate for 15 min.
  • Regeneration buffer 10mM Glycine pH 1.5 (GE Healthcare, BR-1003-54) was run at a flow rate of 10 ⁇ l/min for 30s, and the association rate Ka, dissociation rate Kd, and dissociation constant (ie, affinity K D ) were calculated. See Table 8 and Table 9 for the results.
  • the results of binding to human PD-1 antigen showed that the humanized antibody A17h1_hIgG4 and the subsequent optimized antibodies A17m09_hIgG4, A17m0901_hIgG4, A17m0902_hIgG4 had similar KD values to pembrolizumab.
  • A17h1_hIgG4, A17m09_hIgG4, A17m0901_hIgG4, A17m0902_hIgG4 all had slower dissociation times than pembrolizumab.
  • DC dendritic cells
  • allogeneic T cells can activate T cells and promote T cells to secrete cytokines. This process is inhibited by PD-1/PD-L1 signaling, and the use of PD-1 antibodies can relieve inhibition and promote T cell activation. Therefore, the in vitro drug efficacy of PD-1 antibody can be tested by using DC:T mixed lymphatic reaction experiment.
  • Mononuclear cells were isolated from fresh peripheral blood (PBMC) of a healthy person using a sorting kit (cat: 19359, stemcell). Using a DC cell induction kit (cat: 10985, stemcell), after seven days of culture, monocytes were induced to differentiate into mature DC cells. Then use the sorting kit (cat: 17951, stemcell) to sort out the allogeneic T cells from the fresh PBMC of another healthy person. 5,000 mature DC cells were co-cultured with 50,000 allogeneic T cells, and corresponding concentrations of candidate antibodies were added.
  • PBMC peripheral blood
  • a sorting kit catalog: 19359, stemcell
  • the cell supernatant was aspirated, and the Cisbio-HTRF IFN- ⁇ detection kit was used (Cat: 62HIFNGPEH, Perkinelmer) The concentration of IFN- ⁇ was detected and EC 50 was calculated.
  • A17m09_hIgG4 showed basically the same in vitro efficacy as pembrolizumab in the MLR experiment. Among them, the EC 50 of A17m09_hIgG4 was 0.2931nM, and the EC 50 of pembrolizumab was 0.3271nM.
  • the in vivo anti-tumor efficacy of the PD-1 antibody was verified on the MC38 tumor model of humanized PD-1C57BL/6 mice.
  • the MC38 mouse colon cancer cell line was cultured in DMEM medium (10% FBS), and 5 ⁇ 10 5 MC38 cells were inoculated subcutaneously in humanized PD-1C57BL/6 female mice (Jicui Yaokang). When the volume reached about 80mm 3 , they were randomly divided into groups of 8 mice, given intraperitoneal injection of antibody or control, and the tumor volume was measured twice a week, weighed, and recorded. See Table 10 for experimental grouping and dosing regimen. Considering that the molecular weight of A17m09_hIgG4 is about 75kDa, which is only half of the normal IgG molecular weight, the dosage was adjusted to half of that of pembrolizumab to achieve an equimolar dosage.
  • A17m09_hIgG4 and pembrolizumab have the same antitumor efficacy in vivo as pembrolizumab at the same molar dose.
  • the tumor volume and tumor inhibition rate are shown in Table 11.
  • the tumor inhibition rate of A17m09_hIgG4 is slightly better than that of pembrolizumab at an equimolar dose.
  • Example 8 Construction of fusion protein (PD-1-IL-2v) of anti-PD-1 antibody and IL-2 mutant
  • Wild-type IL-2 preferentially activates regulatory T cells (Tregs) over NK and CD8 + T cells because Tregs highly express IL-2's high-affinity receptor IL-2R ⁇ .
  • Tregs regulatory T cells
  • a common practice is to introduce mutations in IL-2, such as F42A and F72G, to remove the combination of IL-2 and IL-2R ⁇ .
  • IL-2 mutant IL-2v F42A, L72G
  • IL-2 To increase the specificity of IL-2, we introduced the mutation V91T, which reduces the affinity of IL-2 to IL-2R ⁇ , thereby reducing the activity of IL-2 on NK and CD8 + T cells. Further, we brought the IL-2 mutants to PD-1-positive NK and T cells by coupling the above-mentioned IL-2 mutants with anti-PD-1 antibodies, and specifically activated these cells.
  • the fusion protein of anti-PD-1 antibody and IL-2 mutant is constructed as follows according to the schematic diagram of the molecular form shown in Figure 4 .
  • the Fcs of the molecules in Figure 4 all use Knob-in-Hole (KIH) technology, that is, S354C, T366W (Eu numbering system) (constituting the Knob chain) and Y349C, T366S, L368A, Y407V (Eu numbering system) (constituting the Hole chain) to promote the formation of heterodimers; in order to facilitate purification, H435R, Y436F (Eu numbering system) were additionally introduced into the Hole chain; in addition, both Knob and Hole chains were introduced into L234A, L235A and P329G mutations to eliminate the ADCC effect; for heavy chains without light chain pairing, a C220S mutation was introduced in the hinge region to remove free, unpaired cysteine.
  • KH Knob-in-Hole
  • IL-2 uses mutant IL-2v (V91T), which includes 1) T3A to remove possible glycosylation modifications when the protein is expressed in mammalian cells; 2) R38E, and F42A to eliminate IL-2 and Binding of CD25 (i.e., IL-2R ⁇ ); 3) V91T, to reduce IL-2 binding to the intermediate affinity IL-2 receptor IL-2R ⁇ , and 4) C125A, to remove free cysteine from IL-2 Acid, to prevent IL-2 from forming intermolecular disulfide bonds.
  • V91T mutant IL-2v
  • the anti-PD-1 nanobody A17m0902 is coupled to the N-terminals of the Knob and Hole chains respectively through the IgG1 heavy chain hinge region; in the molecule B-V91T shown in Figure 4B , connect the two anti-PD-1 Nanobodies A17m0902 end-to-end through the (G 4 S) 2 linker, and then couple their C-terminals to the N-terminals of the Knob and Hole chains respectively through the IgG1 heavy chain hinge region;
  • Figure 4 In the molecule C-V91T shown in C, the anti-PD-1 Nanobody A17m0902 is coupled to the N-terminal of the Hole chain through the IgG1 heavy chain hinge region, and the heavy chain of the Fab fragment corresponding to the anti-PD-1 antibody pembrolizumab is passed through The IgG1 heavy chain hinge region is coupled to the N-terminal of the Knob chain; in the molecule D-V91T shown in D of Figure 4, two anti-
  • IL-2v (V91T) in the molecules of AE is connected to the C-terminus of the Knob chain of Fc through the (G 4 S) 3 linker, and IL-2v (V91T) in the molecule of F is connected to the Knob chain through the G 4 S linker The N-terminus of the chain is coupled.
  • the Hole chain of C-V91T is SEQ ID NO:44.
  • the Knob chain of D-V91T is SEQ ID NO:47, the Hole chain is SEQ ID NO:46, and the light chain is SEQ ID NO:48.
  • the light chain of E-V91T is SEQ ID NO:48.
  • the Hole chain of F-V91T is SEQ ID NO:46.
  • the IL-2v (V91T) sequence in the above PD-1-IL-2v is:
  • IL-2v(V91T) contains T3A/R38E/F42A/V91T/C125A.
  • the above fusion protein was expressed in HEK293 cells and purified by Protein A.
  • the molecular weight of the purified product was confirmed by SDS-PAGE as expected, which proved that the target protein was prepared.
  • Example 9 Detection of PD-1-IL-2v promoting activity of M07e cell proliferation
  • M07e is a human cell line that only expresses IL-2R ⁇ and IL-2R ⁇ but not IL-2R ⁇ .
  • V91T PD-1-IL-2v
  • Complete medium RPMI 1640+2mM L-glutamine+1mM sodium pyruvate+10% fetal bovine serum+15ng/mL GM-CSF; basal medium: RPMI 1640+2mM L-glutamine+1mM sodium pyruvate +10% fetal bovine serum.
  • M07e cell proliferation experiment M07e cells or M07e-hPD1 were cultured with complete medium at 37°C and 5% CO 2 , and the cells were passaged. After 1-2 days, the cells were collected by centrifugation, washed 3 times with PBS, and resuspended in The cell suspension containing 6.0 ⁇ 10 5 cells per mL was prepared in the basal culture medium, and placed in a 96-well plate with a volume of 50 ⁇ L per well. Add 50 ⁇ L of 2 ⁇ the corresponding concentration of the test drug formulated with the basal culture medium. After the cells have been cultured for 72 hours, add 100 ⁇ L of the lysate to each well. Absorbance was measured with a microplate reader.
  • the proliferative activity data of different forms of PD-1-IL-2v(V91T) fusion proteins on M07e and M07e-hPD-1 cells are shown in Table 12 and Figure 5A, Figure 5B.
  • PD-1-IL-2v(V91T) is less active than PEG-IL-2v in M07e without PD-1 expression, it can still stimulate cell proliferation, indicating that IL-2v(V91T) can still be stronger Despite binding to the IL-2 intermediate affinity receptor IL-2R ⁇ , PD-1-IL-2(V91T) may still systemically activate NK and CD8 + T cells and cause toxicity.
  • All IL-2 mutants have F42A and L72G mutations to eliminate the binding of IL-2 to IL-2R ⁇ ; in addition, for stability reasons, N26Q, N29S and N71Q were added to IL-2 mutants to remove The possible deamidation modification in IL-2 increases the stability of IL-2. And, they also carry their own unique mutations as shown in Table 13, all of which reduce the affinity of IL-2 to the intermediate affinity receptor IL-2R ⁇ to varying degrees.
  • the antibody may contain H435R, Y436F (Eu numbering system) mutations in the Hole chain.
  • the 356th and 358th amino acids of the Hole chain may be D356, L358 or E356, M358 (Eu numbering system), which appear in different alleles and do not affect the function of the antibody.
  • IgG-PC differs from IgG-V91T, IgG-D20A, and IgG-N88D only in part of IL-2.
  • the sequence is as follows:
  • the above fusion protein was expressed in HEK293 cells and purified by Protein A.
  • the molecular weight of the purified product was confirmed by SDS-PAGE as expected, proving that the target protein was obtained.
  • wild-type IL-2 sequence of the present disclosure is:
  • Example 11 Detection of more PD-1-IL-2v promoting activity of M07e cell proliferation
  • Example 10 In order to detect the activity of the above-mentioned PD-1-IL-2v fusion protein with different IL-2 mutants and the dependence on PD-1 expression, we tested the expression in Example 10 in M07e and M07e-hPD-1 cell lines Effects of Fusion Proteins on Cell Proliferation. For the experimental method, see Example 9. The relative cell proliferation activity of PD-1-IL-2v was calculated as follows:
  • PD-1-IL-2v with D20A, D20N, N88R, N88D or Q126D mutations basically None of them activated M07e cells, but they could strongly stimulate the proliferation of M07e-hPD-1 cells, reflecting that PD-1-IL-2v reduced the effect on PD-1 while retaining the selective activation of PD-1 positive cells.
  • Unstimulated T cells did not express or lowly expressed PD-1, while T cells stimulated with CD3 antibody highly expressed PD-1.
  • PBMCs peripheral blood cells
  • T cell proliferation experiment stimulated by CD3 antibody after recovery of cryopreserved human PBMC, T cells were isolated using Pan T Cell Isolation Kit (Miltenyi, 130-096-535) to obtain T cells.
  • the CD3 antibody was diluted to 0.1 ⁇ g/mL with PBS, added to a 10 cm culture dish, incubated at 37°C for 2 hours for coating, washed with PBS, and then used.
  • T cells were plated on 10 cm culture dishes pre-coated with CD3 antibody at 2 ⁇ 10 6 cells/mL, and stimulated for 24 hours at 37°C and 5% CO 2 .
  • Collect the stimulated T cells wash and count, adjust the cell density to 5 ⁇ 10 5 cells/mL, plant 200 ⁇ L per well in a 96-well plate, and dilute the samples to be tested in gradients with medium to make a 5 ⁇ working solution. 50 ⁇ L of the samples to be tested at each concentration were added to the stimulated T cells, mixed evenly, and cultured for 4 days.
  • FACS FACS buffer
  • PBS+2% FBS FACS buffer
  • the cells were labeled with Fixable Viability Dye (eBioscience, 65-0865-14), and tested by CD3 (BD, 565491), CD4 (BD, 558116 ), CD8 (BD, 565310), Ki-67 (eBioscience, 12-5698-82)) after flow-type antibody staining, FACS was used to detect cell proliferation.
  • Unstimulated T cell proliferation experiment After counting the isolated T cells, they were plated in 96-well plates at 5 ⁇ 10 5 cells/mL, 200 ⁇ L per well, and the samples to be tested were serially diluted with medium to make 5 ⁇ working solution, 50 ⁇ L of the samples to be tested at each concentration were added to the stimulated T cells, mixed evenly, and cultured for 4 days. After adding the drug, the cells were washed twice with FACS buffer, and the dead cells were marked with FVD780. After staining with flow cytometry antibodies, the cell proliferation was detected by FACS.
  • each PD-1-IL-2v exhibited significant activity of promoting T cell proliferation at a lower concentration (0.1 nM).
  • the maximal activity of PD-1-IL-2v carrying other mutations on the proliferation of pan T cells stimulated by CD3 antibody was slightly lower.
  • PD-1-IL-2v with D20A, D20N, N88R, N88D, or Q126D mutations were not activated Unstimulated T cells proliferate, but can strongly stimulate the proliferation of T cells stimulated by CD3 antibody; while A-PC and A-V91T-02, although they can more strongly activate the proliferation of T cells stimulated by CD3 antibody , but it can also strongly activate T cells that have not been stimulated by CD3 antibodies.
  • Example 13 Identification of binding affinity between PD-1-IL-2v and antigen PD-1 and IL-2R ⁇ or IL-2R ⁇ / ⁇
  • SPR Surface plasmon resonance
  • the anti-human IgG (Fc) antibody was prepared into a 25 ⁇ g/mL solution with 10 mM sodium acetate buffer (pH 5.0), and the Immobilization program was selected to automatically carry out amino-coupling immobilization of the anti-human IgG (Fc) antibody channel.
  • Each PD-1-IL-2v to be tested was prepared as a ligand with HBS-EP+ buffer solution, and captured with an anti-human IgG (Fc) antibody on the chip channel.
  • Human PD-1 antigen protein (Sino Biological, 10377-H08H), human IL-2R ⁇ receptor protein (Sino Biological, 10165-H08H) or human IL-2R ⁇ / ⁇ receptor heterodimer protein (Acrosystem, ILG- H5283) was used as the analyte, prepared with HBS-EP+ buffer solution, the analyte was serially diluted 2 times from 200nM, a total of 7 concentration points were diluted, and flowed through the experimental channel and the reference channel at a flow rate of 30 ⁇ L/min, and the binding time was 140s , dissociation time 600s.
  • Regeneration buffer 10 mM Glycine pH 1.5 (GE Healthcare, 29238268-AA) was run at a flow rate of 10 ⁇ l/min for 30 s. Calculate the association rate Ka and the dissociation rate Kd, as well as the dissociation constant (ie, the affinity K D value). See Table 18 and Table 19 for results.
  • the results of binding to human PD-1 antigen showed that A-PC, A-N88D and A-D20A had similar binding abilities to human PD-1, and A17m0902_hIgG4 had similar binding abilities to human PD-1 (Table 8).
  • the results of binding to human IL-2R ⁇ showed that A-PC, A-N88D and A-D20A did not bind to human IL-2R ⁇ .
  • the results of binding to human IL-2R ⁇ / ⁇ showed that A-PC had the strongest affinity for human IL-2R ⁇ / ⁇ , and the affinity of A-N88D and A-D20A for human IL-2R ⁇ / ⁇ were respectively reduced by about 13 times and about 63 times.
  • Example 14 Construction and affinity identification of fusion proteins (PD-1-IL-2v) of anti-PD-1 nanobodies with different PD-1 binding affinities and IL-2 mutants
  • nanobodies with different affinities for PD-1 can be obtained.
  • the anti-PD-1 nanobody mutation introduced in A-N88D is shown in Table 21.
  • the anti-PD-1 nanobody sequence after the introduction of the mutation is as follows (the underline is CDR):
  • the A17m09 related antibody has the following general formula sequence:
  • CDR1 may be selected from:
  • CDR2 can be selected from:
  • IISGSGGITHYADSVKG (SEQ ID NO: 95).
  • CDR3 can be selected from:
  • VSDWEDY (SEQ ID NO: 41)
  • A17 of the present disclosure has the following general formula sequence:
  • CDR2 IISGSGX 1 X 2 X 3 HYX 7 DSVKG, wherein X 1 is selected from V or G, X 2 is selected from I or S, X 3 is selected from T or A, X 7 is selected from V or A (SEQ ID NO: 100)
  • CDR3 VSDWX 9 X 5 Y, wherein, X 9 is selected from Q, D or E, and X 5 is selected from D or E (SEQ ID NO: 101)
  • CDR1 may be selected from:
  • CDR2 can be selected from:
  • IISGSGGITHYADSVKG (SEQ ID NO: 95).
  • CDR3 can be:
  • VSDWDDY (SEQ ID NO: 9)
  • VSDWEDY (SEQ ID NO: 41)
  • the fusion proteins (PD-1-IL-2v) of anti-PD-1 nanobody mutants and IL-2 mutants are A-N88D-M08, A-N88D-M15, A-N88D-M26, A-N88D- M27, A-N88D-M28.
  • A-N88D-M08 the full-length sequence of A-N88D-M08 is given, Fc is underlined, and linker is italicized.
  • Table 23 SPR affinity data for PD-1 antigen of PD-1-IL-2v containing different anti-PD-1 Nanobody mutants The results in Table 23 show that all mutants can effectively bind to human PD-1.
  • Example 15 Detection of the M07e cell proliferation-promoting activity of the fusion protein (PD-1-IL-2v) of anti-PD-1 antibodies and IL-2 mutants with different PD-1 binding affinities
  • Example 14 In order to detect the activity of PD-1-IL-2v fusion proteins with different PD-1 binding affinities and the dependence on PD-1 binding affinities, we tested the M07e-hPD-1 cell lines mentioned in Example 14 Effect of fusion protein of PD-1 antibody mutant and IL-2v on cell proliferation. For the experimental method and the calculation method of the relative cell proliferation activity of PD-1-IL-2v, see Example 11.
  • the experimental results are shown in Table 24 and Figure 10.
  • the results showed that A-N88D-M08, A-N88D-M15, A-N88D-M26, A-N88D-M27 and A-N88D-M28 all had the activity of promoting the proliferation of M07e-hPD-1 cells.
  • the PD-1-IL-2v fusion protein promotes the proliferation activity of M07e-hPD-1 cells and the affinity of binding to PD-1 antigen (Table 23) presents a basically consistent trend, indicating that the PD-1-IL-2v fusion protein promotes cell proliferation activity The strength depends on the strength of the binding affinity of PD-1-IL-2v to PD-1.
  • Example 16 A-N88D can inhibit the growth of mouse CT26 tumor
  • the in vivo anti-tumor efficacy of A-N88D constructed in Example 10 was verified on the CT26 tumor model of humanized PD-1Balb/c mice.
  • the CT26 mouse colon cancer cell line was cultured in DMEM medium (10% FBS), and 5 ⁇ 10 6 CT26 cells were inoculated subcutaneously in humanized PD-1Balb/c female mice (purchased from Jizui Yaokang).
  • DMEM medium 10% FBS
  • 5 ⁇ 10 6 CT26 cells were inoculated subcutaneously in humanized PD-1Balb/c female mice (purchased from Jizui Yaokang).
  • the dosage of A-N88D is adjusted to 120% of that of A17m0902_hIgG4 to achieve equimolar dosage.
  • Example 17 A-N88D can inhibit the growth of mouse B16F10 tumor
  • the in vivo anti-tumor efficacy of A-N88D was verified on the B16F10 tumor model of humanized PD-1C57BL/6 mice.
  • the B16F10 mouse skin melanoma cell line was cultured in DMEM medium (10% FBS), and 1 ⁇ 10 5 B16F10 cells were inoculated subcutaneously in humanized PD-1C57BL/6 female mice (purchased from Biocytogen) and waited for small
  • the average tumor volume of the mice reached about 75mm 3 , they were randomly divided into groups of 8 mice, and given intraperitoneal injection of PBS and A-N88D (24mg/kg). The tumor volume was measured every two days, and the body weight was weighed and the data was recorded.

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Abstract

La présente divulgation concerne un immunoconjugué et son utilisation. En particulier, la présente divulgation concerne un immunoconjugué comprenant un anticorps PD-1 ou un fragment de liaison à l'antigène de celui-ci et IL-2, et l'utilisation de l'immunoconjugué pour le traitement du cancer.
PCT/CN2023/075448 2022-02-11 2023-02-10 Immunoconjugué et son utilisation WO2023151661A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110382525A (zh) * 2017-04-03 2019-10-25 豪夫迈·罗氏有限公司 免疫缀合物
CN110392692A (zh) * 2017-04-03 2019-10-29 豪夫迈·罗氏有限公司 抗pd-1抗体与突变体il-2或与il-15的免疫缀合物
CN111647068A (zh) * 2019-03-04 2020-09-11 江苏恒瑞医药股份有限公司 一种人白细胞介素2变体或其衍生物
US20210260163A1 (en) * 2018-03-09 2021-08-26 AskGene Pharma, Inc. Novel cytokine prodrugs
WO2021255138A1 (fr) * 2020-06-19 2021-12-23 F. Hoffmann-La Roche Ag Molécules de liaison au domaine fc activant l'immunité
WO2022006380A2 (fr) * 2020-07-02 2022-01-06 Inhibrx, Inc. Polypeptides comprenant des polypeptides à il-2 modifiée et leurs utilisations

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110382525A (zh) * 2017-04-03 2019-10-25 豪夫迈·罗氏有限公司 免疫缀合物
CN110392692A (zh) * 2017-04-03 2019-10-29 豪夫迈·罗氏有限公司 抗pd-1抗体与突变体il-2或与il-15的免疫缀合物
US20210260163A1 (en) * 2018-03-09 2021-08-26 AskGene Pharma, Inc. Novel cytokine prodrugs
CN111647068A (zh) * 2019-03-04 2020-09-11 江苏恒瑞医药股份有限公司 一种人白细胞介素2变体或其衍生物
WO2021255138A1 (fr) * 2020-06-19 2021-12-23 F. Hoffmann-La Roche Ag Molécules de liaison au domaine fc activant l'immunité
WO2022006380A2 (fr) * 2020-07-02 2022-01-06 Inhibrx, Inc. Polypeptides comprenant des polypeptides à il-2 modifiée et leurs utilisations

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